forenseq universal analysis software v2.0 reference guide · index cv displayed as a percentage,...

ForenSeq™ Universal Analysis Software v2.0

Reference Guide

VEROGEN PROPRIETARY Document # VD2019002 Rev. A

August 2019

ForenSeq Universal Analysis Software v2.0 Reference Guide Document # VD2019002 Rev. A 2

This document and its contents are proprietary to Verogen, Inc. and its affiliate s, and are intended solely for the

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purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise

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Verogen does not convey any license under its patent, trademark, copyright, or common-law rights nor similar

rights of any third parties by this document.

The instructions in this document must be strictly and explicitly followed by qualified and properly trained

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FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED

HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR

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© 2019 Verogen, Inc. All rights reserved.

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For Research, Forensic, or Paternity Use Only. Not for any clinical or therapeutic use in humans or animals.


Table of Contents

Revision History ....................................................................................................... 5

Getting Started ..................................................................... 6 Introduction .............................................................................................................. 7 Software Functions .................................................................................................. 8 ForenSeq Universal Analysis Software Concepts ................................................... 9 Viewing the ForenSeq Universal Analysis Software .............................................. 15 Logging into the Software ...................................................................................... 16 Viewing the System Dashboard ............................................................................. 19 Creating a New Run ............................................................................................... 20 Viewing Run Details ............................................................................................... 26 Modifying Run Details ............................................................................................ 28 Viewing Quality Metrics.......................................................................................... 31

Sample and Run Results ................................................... 37 Introduction ............................................................................................................ 38 Viewing a Project ................................................................................................... 39 Viewing Quality and Control Metrics ...................................................................... 43 Viewing Sample Details ......................................................................................... 48 Adding Samples to a Project ................................................................................. 57 Reanalyzing Samples ............................................................................................ 58

Sample Comparison .......................................................... 60 Introduction ............................................................................................................ 61 Sample Comparison .............................................................................................. 62

Reports .............................................................................. 67 Introduction ............................................................................................................ 68 Project Report ........................................................................................................ 69 Sample Report ....................................................................................................... 72 CODIS Report ........................................................................................................ 75 EMPOP Report ...................................................................................................... 81

System Settings ................................................................. 84 Introduction ............................................................................................................ 85 Analysis Methods ................................................................................................... 86 CODIS Report Defaults.......................................................................................... 97 User Management ............................................................................................... 101

Troubleshooting ............................................................... 104 Introduction .......................................................................................................... 105 Troubleshooting ForenSeq Runs ......................................................................... 106 Troubleshooting Analysis Errors .......................................................................... 109


Appendix A Supporting Information .................................................. 110 Base Call Codes .................................................................................................. 111 Human Sequencing Control Loci ......................................................................... 113 Nuclear mtDNA Insertions (NUMTs) .................................................................... 114 Coverage of the HVII C-stretch ............................................................................ 115


Revision History

Document # Date Description of Change

VD2019002 Rev. A August 2019 Initial release.

ForenSeq Universal Analysis Software v2.0 Reference Guide

Document # VD2019002 Rev. A 6

Getting Started

Introduction ..............................................................................................................7 Software Functions ..................................................................................................8 ForenSeq Universal Analysis Software Concepts ...................................................9 Viewing the ForenSeq Universal Analysis Software ............................................. 15 Logging into the Software ..................................................................................... 16

Creating an Account ........................................................................................ 16 Logging In ........................................................................................................ 17 Logging Out ..................................................................................................... 17

Viewing the System Dashboard ............................................................................ 19 Sidebar Menu .................................................................................................. 19

Creating a New Run .............................................................................................. 20 Flow Cell Types ............................................................................................... 20 Create a Run ................................................................................................... 20 Entering Sample Information ........................................................................... 22

Importing Samples ...................................................................................... 22 Adding Samples Individually ...................................................................... 24

Viewing Run Details .............................................................................................. 26 Viewing Run Status ......................................................................................... 26 Searching Samples .......................................................................................... 26 Filtering Samples ............................................................................................. 27

Modifying Run Details ........................................................................................... 28 Editing a Run ................................................................................................... 28 Editing a Sample .............................................................................................. 28 Removing a Sample ........................................................................................ 29

Viewing Quality Metrics ......................................................................................... 31 Quality Metrics ................................................................................................. 31

Run Quality Metrics .................................................................................... 31 Reads and Index Quality Metrics: ForenSeq mtDNA Control Region Kit .. 32 Human Sequencing Control ....................................................................... 33

Sample Representation ................................................................................... 34 Index CV ..................................................................................................... 35 Sample Representation Read Count Guideline ......................................... 35



Introduction

ForenSeq™ Universal Analysis Software is a DNA-to-data forensic software solution and is a

part of the MiSeq FGx™ Forensic Genomics System. The software performs analysis of

sequenced genetic Sample information and works in combination with the ForenSeq mtDNA

Control Region Kit and the MiSeq FGx instrument.

The ForenSeq Universal Analysis Software comes pre-installed as a dedicated server with a

user interface specific for mtDNA forensic genomics, and enables Run setup, Sample

management, analysis, and report generation.

NOTE

Some laboratories may choose to or need to perform internal validation studies in order to

develop interpretation guidelines for casework and DNA databasing using the ForenSeq

Universal Analysis Software. It is possible that some features and recommendations detailed

in the guide are not part of your analysis requirements, analysis routine, or necessary for

high-quality analyses.



Software Functions

The following is a workflow outline of functions administered by the software:

1. Create an account with a user name and password for access to the ForenSeq

Universal Analysis Software and the MiSeq FGx instrument.

2. Create a Run. Input Sample index combinations, Sample types. Enter information

manually or import a text (*.txt) file.

3. The MiSeq FGx uses two software applications in tandem to produce images of

clusters on the flow cell, perform image analysis, and call bases.

a. During the Run, MiSeq FGx Control Software captures images of clusters on the

flow cell for image analysis, as well as operates the flow cell stage, gives

commands to dispense reagents, and changes temperatures of the flow cell.

b. Real-Time Analysis (RTA) software performs image analysis, base calling, and

assigns a quality score to each base for each cycle as the Run progresses. The

completion of analysis by RTA and transfer of files initiates analysis on the

ForenSeq Universal Analysis Software.

4. Using data from RTA, the ForenSeq Universal Analysis Software aligns reads to make

variant calls. The ForenSeq Universal Analysis Software provides quality indicators

intended to assist when interpretation or troubleshooting might be considered.

5. Review Run analyses and generate reports in the ForenSeq Universal Analysis

Software.



ForenSeq Universal Analysis Software Concepts

The following concepts and terms apply to the ForenSeq Universal Analysis Software:

Table 1 Concepts and Terms

Concept Description

Action Provides option(s) such as editing Sample information or downloading

Sample, Project or Reports.

Amplicon The product of PCR amplification of a targeted region of interest from

input DNA template.

Amplicon End

Coordinate

Last nucleotide position of a targeted mtDNA amplicon, inclusive of PCR

primer sequence. Together, the Amplicon Start Coordinate and the

Amplicon End Coordinate provide the coordinate range of nucleotides

for each targeted amplicon.

Amplicon Start

Coordinate

First nucleotide position of a targeted mtDNA amplicon, inclusive of

PCR primer sequence. Together, the Amplicon Start Coordinate and the

Amplicon End Coordinate provide the coordinate range of nucleotides

for each targeted amplicon.

Analysis Method A collection of settings and thresholds used to inform the analysis of

sequencing data to generate allele or variant calls. A collection of settings

and thresholds used to inform the analysis of sequencing data to

generate allele or variant calls.

Analysis State Sample Analysis States are as follows:

New: Analysis by the software has not yet begun

Queued: Analysis is in line to begin processing

Processing: Analysis by the software is in progress

Completed: Analysis by the software completed

successfully

Errored: Analysis did not complete successfully

Analytical

Threshold

If the read count for a mtDNA nucleotide call is less than the value of the

user-adjustable Analytical Threshold (%), then the nucleotide call is not

visible in the UI nor reported as part of the call.

Assay Type The ForenSeq kit used to prepare a Sample in preparation for sequencing

and for further analysis. For mtDNA, a custom assay type may be

selected.

Call The reported nucleotide (base) for the position (coordinate), based on an

Analysis Method.

Cluster Density

(K/mm²)

Shows the number of clusters per square millimeter for the Run. For

ForenSeq Runs, the target cluster density range is 400–1650 K/mm².

Cluster density values outside of the target range can still produce results

that are sufficient to use for analysis. Values that deviate substantially

from the target range can negatively impact other quality metrics and

decrease the quantity of valuable data from the Run.

Control Region The range positions from 16024 to 576 for mtDNA.



Concept Description

Coverage Coverage describes the average number of reads, for a locus, an allele or

a variant position in a Sample, that align to or "cover" known reference

bases (e.g., relative to rCRS). Sequencing coverage level (read depth) can

help to determine whether or not an allele call or variant call can be made

with confidence.

Clusters Passing

Filter (%)

Shows the percentage of clusters passing filter based on the Illumina

chastity filter1, which measures quality. The filter can detect low quality

base calls. Data appears in the UI after cycle 25.

Cycle The number of completed sequencing cycles is displayed on the Run

Details page. One cycle includes the chemical addition and imaging of

one base for each cluster on a flow cell. The ForenSeq mtDNA Control

Region Assay Type consists of 318 cycles. The mtDNA Custom Assay

Type instructs the number of cycles based on the library Type contained

in an Analysis Method: Non-Directed Sequencing results in 318 cycles

and Primer-Directed Sequencing Results in 618 cycles.

Deletions The number of one or more nucleotides deleted from the Sample mtDNA

in comparison to the rCRS.

Description The pertinent information for each Run or Sample as defined by the user.

Differences A call that differs between Samples when compared using the Sample

Compare feature.

Differences

Indicator

If there are different allele or varinats calls among Samples, a differences

indicator appears when using the Sample Compare feature.

Flow Cell Type MiSeq FGx Reagent Kits and MiSeq FGx Reagent Micro Kits are available

for use with ForenSeq kits. The capacity of the flow cells in each type of

kits varies. The MiSeq FGx Standard flow cell type allows for more

Samples to be sequenced simultaneously as compared to a MiSeq FGx

Micro flow cell. Both flow cell types use the same sequencing reactions

for equivalent data quality.

Forward Primer

Length (bases)

The number of bases (nucleotides) that comprise your forward PCR

primer (oligonucleotide) for mtDNA amplicons.

Forward Primer

Start Coordinate

The start coordinate (5' end) for your forward PCR primer for mtDNA

amplicons.

For example, the Verogen mtDNA Custom Analysis Method contains a

Forward Primer Start Coordinate of 9397 for amplicon MTL F1 – MTL R1

and a Forward Primer Start Coordinate of 15195 for amplicon MTL F2 –

MTL R2.

Hotspot A hotspot in a selected Sample is a position where one or more of the

following are indicated: a variant, interpretation threshold indicator,

differences indicator, no call indicator, call indicator or user modification.

Hotspots are indicated by the large navigation arrows found at the top of

the Position Viewer.

1 Calculating Percent Passing Filter for Patterned and Nonpatterned Flow Cells. (2017).

[ebook] San Diego: Illumina. Available at: https://www.illumina.com/content/dam/illumina-

marketing/documents/products/technotes/hiseq-x-percent-pf-technical-note-770-2014-043.pdf



Concept Description

Human Sequencing

Control

Pool of human DNA STR loci used as a positive control for the

sequencing portion of processing Samples using a ForenSeq kit. Human

Sequencing Control (HSC) helps MiSeq FGx Run completion and may

assist in troubleshooting, if needed.

Index DNA tags that are attached to DNA sequences targeted by the ForenSeq

mtDNA Control RegionKit, enabling multiple Samples to be pooled

together for sequencing and demultiplexed post-Run.

Index CV Displayed as a percentage, the number of reads that are assigned to each

Sample. CV is the coefficient of variation for the number of read counts

across all indexes. Index CV represents the distribution of read counts of

the Samples in the Run.

Insertions The number of one or more nucleotides inserted in the Sample mtDNA

genome in comparison to a reference genome (rCRS).

Interpretation

Threshold

Defined in an Analysis Method, a nucleotide with total number of reads

greater than or equal to the interpretation threshold contributes to the

call for the mtDNA coordinate. When more than one base exceeds this

threshold for a coordinate, the IUPAC code for the bases is reported.

Interpretation

Threshold Indicator

If the read count of a mtDNA sequence has a read count between the

analytical and interpretation threshold, an interpretation threshold

indicator icon appears in the UI.

Library Type When using an Analysis Method with data from a custom mtDNA assay,

two options for Library Type are available: Primer-Directed Sequencing,

where one strand is always sequenced as Read 1 and the opposite strand

is always sequenced as Read 2, and Non-Directed Sequencing where

either strand may be sequenced as Read 1 or as Read 2 (e.g., libraries

created using Nextera XT DNA Library Prep Kits). Library Type must be

user-selected for custom mtDNA analysis when creating an Analysis

Method and does not need to be selected for the ForenSeq Control

Region Kit.

Minimum Quality

Score

Defined in an Analysis Method, reads at or above the Q score can

contribute to the variant call at each mtDNA coordinate.

Minimum Read

Count

Defined in an Analysis Method, the Minimum Read Count must be

reached at a position for a nucleotide in order to be considered for base

calling.

N Indicator Quality indicator for reagent blanks and negative amplification control

Samples.

Name The file designation assigned to each Run, Sample, and Project as defined

by the user.

Negative

Amplification

Control

A Sample where DNA was not added as PCR template for amplification.

No Call At a position, if no read counts for a specific nucleotide (A, C, G or T)

meet or exceed the interpretation threshold, then a No Call is assigned.

No Call Indicator If a position does not contain a call, a No Call indicator appears in the

Position Viewer.



Concept Description

Original Call An mtDNA variant call made based on the Run Analysis Method,

without user-modification.

P Indicator Quality indicator for positive amplification controls that use positive

control DNA provided in the ForenSeq kit.

Page A page is a display of 15 positions in the Position Viewer. Navigate

across pages using the small arrows found at the top of the Position

Viewer.

Percent A, C, G, T The percentage of forward and reverse reads out of the total reads for the

A, C, G, or T bases at the call position in mtDNA.

Percent Del The percentage of forward and reverse reads out of the total reads for a

deletion at the call position in mtDNA.

Percent Ref The percentage of forward and reverse reads out of the total reads that

don’t support an insertion at an insertion point in mtDNA.

Percentages The percentages of each nucleotide called at a position are stacked and

shown in relation to the thresholds. Percentages are displayed in the

details of a selected position in the Position Viewer.

Phasing (%) Shows the percentage of molecules in a cluster that fall behind the

current cycle within Read 1 and Read 2. Lower percentages are indicative

of higher quality Run statistics. For ForenSeq Samples, a phasing value of

≤ 0.25% is recommended. Phasing values outside of the target range can

still produce results that are sufficient to use for analysis.

Sample Plate The 96-well PCR plate in which the Sample library was stored prior to

MiSeq FGx sequencing. For ForenSeq mtDNA library prep kits, this is the

Normalized Library Plate (NLP).

Position Describes a base (nucleotide) coordinate of mtDNA using the numbering

convention of the rCRS, comprised of positions 1 to 16569.

ForenSeq Positive

Amplification

Control

A library of the positive control HL60 DNA provided in the ForenSeq kit.

Prephasing (%) Shows the percentage of molecules in a cluster that Run ahead of the

current cycle within Read 1 and Read 2. Lower percentages are indicative

of higher quality Run statistics. For ForenSeq Samples, a prephasing

value of ≤ 0.15% is recommended. Prephasing values outside of the target

range can still produce results that are sufficient to use for analysis.

Project A Project is a collection of analyzed results for one or more Samples and

is the primary place for viewing and modifying results and creating

Reports. Projects can be used to logically group and organize data. If

desired, each Sample in a Run can be independently assigned to a

different Project, so that a Run can have Samples from more than one

Project. Samples may be assigned to multiple Projects.

Q Indicator Quality metrics indicator for a Run.

rCRS The Revised Cambridge Reference Sequence (rCRS) is used as the

mtDNA reference genome for variant calling.



Concept Description

Read The sequence string of A, T, C, and G bases corresponding to a ForenSeq

amplicon from mtDNA. Millions of reads can be generated in a

sequencing Run from multiple loci and multiple Samples

simultaneously.

Reads A, C, G, T The number of forward and reverse reads for the A, C, G, or T bases at

the mtDNA call position.

Reads Del The number of forward and reverse reads for a deletion at the mtDNA

call position.

Reads Ref The number of forward and reverse reads that don’t support an insertion

at an insertion point in mtDNA.

Reagent Blank A specific type of negative control that may be carried through the

entirety of Sample processing and where DNA is not added as PCR

template for amplification.

Sample of Interest A user-designated Sample for comparison against other Samples when

using the Sample Compare feature.

Read 1 (i5) Primer

Sequence

The base (nucleotide) sequence of the PCR primer that’s tagged with

Illumina’s i5 index. The i5 index is part of the adapter sequence, along

with the binding site for the Read 1 sequencing primer, which drives

Read 1 on the flow cell.

Read 2 (i7) Primer

Sequence

The base (nucleotide) sequence of the PCR primer that’s tagged with

Illumina’s i7 index. The i7 index is part of the adapter sequence, along

with the binding site for the Read 2 sequencing primer, which drives

Read 2 on the flow cell.

Reagent Cartridge

Kit Lot

Refers to component of a MiSeq FGx Reagent Kit or MiSeq FGx Reagent

Micro Kit.

Reanalysis To apply an Analysis Method to a Sample, subsequent to its original Run

Analysis Method. For example, user-adjustable Analysis Method

parameters of ForenSeq mtDNA Control Region Kit are Analytical

Threshold, Interpretation Threshold, Minimum Quality Score and

Minimum Read Count.

Reference Call The base call of the reference genome (rCRS) at a position on the

mitochondrial DNA molecule.

Reverse Primer

Length (bases)

The number of bases (nucleotides) that comprise your reverse PCR

primer (oligonucleotide) for mtDNA amplicons.

Reverse Primer

Start Coordinate

The start coordinate (5' end) for your reverse PCR primer for mtDNA

amplicons.

For example, the Verogen mtDNA Custom Analysis Method contains a

Reverse Primer Start Coordinate of 1892 for amplicon MTL F1 – MTL R1

and a Reverse Primer Start Coordinate of 9796 for amplicon MTL F2 –

MTL R2.

Run Analysis

Method

The Analysis Method assigned to a Sample, on the Run page (the original

Analysis Method prior to possible subsequent reanalysis).

Run States Shows the stage of progress for each Run. (Created, Errored, Sequencing,

Completed, Paused, Stopped)



Concept Description

Runs An independent sequencing event for a collection of Samples.

Sample Biological specimen used in sequencing.

Sample Compare A user-defined tool that examines a selected Sample of Interest against

other Samples.

Sample

Representation

The analyzed intensity (number of reads) per Sample for an entire Run.

These data are available after sequencing and analysis are complete.

Sample Type The type or function of a Sample. Sample types include Sample, positive

amplification control, negative amplification control and reagent blank.

Single Nucleotide

Variants (SNVs)

The number of positions where the mtDNA call for the Sample differs

from the reference genome (rCRS).

Strand Depth Read count from the strand (forward or reverse) that has the majority of

reads for a mtDNA base (nucleotide) position (coordinate).

Strand Direction The selection of a forward or reverse strand when a Primer-Directed

Library type is selected for a Custom Analysis Method.

Total Read Count Total number of reads, from both the forward and reverse DNA strands,

for a mtDNA base (nucleotide) position (coordinate).

User-Modified Call An mtDNA call that was modified by a user, which is presented as

[position] [original call] -> [new call] at the modified position in the

Position Viewer.

User-Modified

Indicator

If the mtDNA base call is manually updated, a user-modified indicator

appears at the modified position in the Position Viewer.

Variant An mtDNA call that differs from the reference genome (rCRS) as

displayed in the Position Viewer.

Variant Indicator If the mtDNA call for a Sample is different from the rCRS base call, a

variant indicator appears at the variant position in the Position Viewer.

Sample Well

Position

The 96-well PCR plate column and row position where a library was

stored prior to pooling, denaturation and loading onto the MiSeq FGx.

For example, the position of the Sample library or control in the upper

left corner of a plate is at position A1, where A is the Row position and 1

is the Column position.



Viewing the ForenSeq Universal Analysis Software

The ForenSeq Universal Analysis Software interface is viewed through a web browser. To

view the interface, open a web browser on a computer with access to the network used by the

ForenSeq Universal Analysis Software and the MiSeq FGx instrument. Set screen resolution to

a minimum of 992 pixels for optimal display.

NOTE

The application is optimized for use on the Google Chrome Browser.

To access the ForenSeq Universal Analysis Software, enter the server address in the web

browser address bar. If the server is not listed in the Domain Name System (DNS), enter the

server IP address of the network.

NOTE

No internet connection or internet access is required to view the software interface.



Logging into the Software

Creating an Account

Prior to accessing the ForenSeq Universal Analysis Software, create an account.

1. Access the network server through a web browser.

A login page appears.

Figure 1-1 Login

2. When the Login page opens, select Register as a New User?

A Register page appears.

Figure 1-2 Register New User

3. Enter an email address.

4. Enter a password.

5. Confirm password.



6. Select Register.

Figure 1-3 Account Registration

7. After your accounted is created, a user with administrator-level system access must

approve your account before you can access the system. See Enabling and Disabling

Accounts.

NOTE

The first user created will automatically be assigned administrator privileges and will be

responsible for enabling any new accounts. There is no password recovery mechanism. If the

administrator password is lost and no secondary administrator account was created, you will

be unable to create any additional administrators or users and the system will require a reset.

It is highly recommended to create a backup administrator account.

Logging In

1. Access the network server through a web browser.

2. Enter a username.

3. Enter a password.

If you need an account, see Creating an Account.

4. Select Login.

The ForenSeq Universal Analysis Software System Dashboard opens.

Logging Out

1. From the ForenSeq Universal Analysis Software System Dashboard, select Account

from the sidebar menu.



Figure 1-4 Logout

2. Select Logout.

A Logout screen appears.

Return to the Login screen by selecting the hyperlinked here on the Logout screen.

Figure 1-5 Logout Screen



Viewing the System Dashboard

A successful login opens the system dashboard. The system dashboard shows information

and functionality organized within Runs and Projects. In the left-hand sidebar menu, the

following information can be accessed:

Home

Runs

Projects

Settings

Account

Access to Runs and Projects is expedited in the system dashboard whether searching for a past

event or creating a new one. Creating a new Run or Project is designed into the system

dashboard so that users can initiate action immediately upon opening the software.

Sidebar Menu

From the sidebar, Home, Runs, Projects, Settings and Account are accessible.

1. Select Home, Runs, Projects, Settings or Account.

The selection pane will expand.

To collapse the selection pane, select the X.

2. Select, Search or Create from the selection pane.

Figure 1-6 System Dashboard



Creating a New Run

The Runs workspace is where data analysis preparation occurs prior to starting a MiSeq FGx

sequencing Run. On the Runs page, Sample information is input, organized, and available for

editing prior to sequencing. Upon initiation of sequencing, Runs provides progress and access

to Sample information. After sequencing is complete, Run Quality Metrics and Sample

Representation are specified for each Run.

Before performing a sequencing Run on the MiSeq FGx instrument, enter information in the

ForenSeq Universal Analysis Software about the Run and the Samples in the Run.

When creating a new Run, select either a Standard or Micro flow cell type. The flow cell is

provided in the MiSeq FGx Reagent kit (standard flow cell) and in the MiSeq FGx Reagent

Micro kit (micro flow cell). Your decision of which flow cell type to use may be based

primarily on the total number of Samples per Run and the desired read coverage level for each

Sample.

Flow Cell Types

Two types of MiSeq FGx Reagent Kits are available. The standard MiSeq FGx Reagent Kit

provides the standard flow cell type and the MiSeq FGx Reagent Micro Kit provides the micro

flow cell type.

Table 2 Flow Cell Types

Flow Cell Types Description

Standard ForenSeq mtDNA Control Region: up to 64 Samples per MiSeq FGx Run when

using the default ForenSeq mtDNA Control Region Analysis Method.

Micro ForenSeq mtDNA Control Region: up to 48 Samples per MiSeq FGx Run when

using the default ForenSeq mtDNA Control Region Analysis Method.

Create a Run

After logging in, the software opens to the system dashboard. The software enables Run

creation directly from the system dashboard.

Figure 1-7 Create Run from System Dashboard

1. Select Create Run.

A Create Run dialogue box appears.



Figure 1-8 Create Run Dialogue

2. Enter the following information:

Run Name

Description

Flow Cell Type

Reagent Cartridge Kit Lot #

NOTE

Fields marked with an asterisk (*) are required information.

3. Select Save.

The Run Details page appears.

Figure 1-9 Run Details



Entering Sample Information

Sample information can be entered into a created Run either by importing a Sample Sheet or

individually adding Samples.

Importing Samples

1. Select Import Samples.

2. Select Drop Files Here or Click to Select File.

Figure 1-10 Importing Samples

3. Navigate to the Sample sheet (*.txt) file and select Open on your computer.

The name of the uploaded Sample sheet file appears in bold below the dashed-line

box.

NOTE

If necessary, select SAMPLE SHEET TEMPLATE to download an example of a

.txt file categorizing Samples and associated information for import.



Figure 1-11 Navigate to Sample Sheet

NOTE

Only one *.txt file can be uploaded for a Run. Attempts to upload another *.txt file

results in an override of the original file.

4. Select Next.

A Review Samples dialog box appears that lists data from the uploaded *.txt file.

Scroll through the list to ensure accuracy.

Figure 1-12 Review Samples

5. Select Import.

Upon completion of Sample(s) import, the Run Details page appears with the

imported Samples.



Figure 1-13 Imported Samples in Run Details

Adding Samples Individually

1. Select Add Sample.

A dialogue box will appear.

Figure 1-14 Add Sample

2. Enter the following information in the dialogue box:

Sample Name

Description

Sample Type (from dropdown box)



Assay Type (from dropdown box)

Index 1 (from dropdown box)

Index 2 (from dropdown box)

Run Analysis Method (from dropdown box)

Initial Project(s)

Sample Plate Name (optional)

Sample Please Well Position (optional)

NOTE


3. Select Save.

Upon saving, the Run Details page appears with the added Sample.



Viewing Run Details

To view the Run Details page, select the name of the Run on the left-hand system dashboard.

There are several ways to find a Run on the system dashboard:

The system dashboard lists Runs arranged by Run creation date with the most

recent Run at the top of the list.

On the system dashboard, use page navigation at the bottom of the Run list to

locate the Run.

On the system dashboard, search by Run Name or Run Description.

The Run Details page contains the following Run information:

Name of Run

Description of Run (if an optional description was included)

State of the Run

(MiSeq FGx) Flow Cell Type (Standard or Micro)

Cycle Count

(MiSeq FGx) Reagent Cartridge Kit Lot Number

Date and time the Run was created in the ForenSeq Universal Analysis Software

Date and time the sequencing completed on the MiSeq FGx, if applicable

Quality metrics for the Run

Sample Representation for the Run

A list of Samples on the Run

Viewing Run Status

After a Run is created and saved, the Run State is indicated under the Run Name in Run

Details.

Table 3 MiSeq FGx Run Status Descriptions

Run Status Description

Created Run setup information is saved in the ForenSeq Universal Analysis

Software.

Sequencing Sequencing of the Run on the MiSeq FGx instrument is in progress.

Completed Sequencing of the Run on the MiSeq FGx instrument is complete.

Errored The Run did not finish sequencing on the MiSeq FGx instrument because

it was manually stopped or because of an error.

Paused Sequencing of the Run is on pause on the MiSeq FGx instrument.

Stopped Sequencing of the Run is stopped on the MiSeq FGx instrument.

Searching Samples

The Search tool filters Run Details according to specific parameters.



1. Click inside the Search bar.

Figure 1-15 Search Bar

2. Enter a Sample Name, Description, Index, or Run Analysis Method.

The Search bar filters results as necessary.

Filtering Samples

The Filter tool sorts groups of data according to specific parameters.

1. Selelct filter icon in either the Sample Type or Assay Type columns.

2. Select an option from the list by selecting a specific box.

3. Select the filter icon.

The filter returns results as necessary.

Figure 1-16 Sample Type Filters

Figure 1-17 Assay Type Filters



Modifying Run Details

Editing a Run

1. Select the pencil icon next to the Run Name to start edit mode.

A dialog box appears with the same fields used when the Run was created: Run

Name, Description, Flow Cell Type (Standard or Micro), and (MiSeq FGx) Reagent

Cartridge Kit Lot Number.

2. Select each field that requires editing and make the necessary changes.

NOTE

Run Name, Description and Reagent Cartridge Kit Lot Number can be

edited any time. Flow Cell Type can only be edited when the Run State

is listed as Created.

3. Select Save.

Figure 1-18 Editing a Run

Editing a Sample

1. Select the pencil icon under the Actions column for the corresponding Sample.

A dialogue box appears that lists Sample Name, Description, Sample Type, Assay

Type, Index 1, Index 2, Run Analysis Method, Initial Projects, Sample Plate and

Sample Well Position.



Figure 1-19 Editing a Sample

NOTE

Assay Type, Index 1, Index 2, Run Analysis Method and Initial Projects fields can only be

edited when the Run State is listed as Created. To edit these fields when the Run State is

listed as Completed, Remove the Sample from the Run, make the necessary changes and

Add the Sample back to the Run.

2.


3. Edit the Initial Projects field as follows:

To add a Sample to a Project, enter the Project name directly inside the box.

To remove a Sample from a Project, select the X next to the Project name.

NOTE

Projects can be added or deleted from the Run page only when the Run State is listed as

Created.

4. Select Save.

Removing a Sample

1. Select the checkbox next to each Sample name to be removed.

The Remove Sample(s) button is active only if at least one Sample is selected.

2. Select Remove Sample(s).

A confirmation box appears with a list of selected Samples.

3. From the confirmation box, select Confirm.



Figure 1-20 Removing Samples



Viewing Quality Metrics

Quality Metrics

To view overall Run quality, select the Quality Metrics indicator in Run Details. A dialogue

box appears displaying Run Quality Metrics, Read and Index Quality Metrics, Cycle Count

and the Human Sequencing Control.

This page enables remote monitoring of the Run and the Quality Metrics during sequencing

on the MiSeq FGx instrument. The metrics presented mirror the information displayed on the

MiSeq FGx instrument during sequencing. After sequencing of the Run completes, the

information for the Run is preserved for Run quality assessment, as the Quality Metrics page

remains static so that results from the Run are available.

A color indicator displays the overall outcome of the quality metrics. Horizontal bars show the

preferred range of values for cluster density, clusters passing filter, phasing, and prephasing.

Read and Index quality and Human Sequencing Control quality is indicated with a short

vertical color bar and corresponding icons.

Figure 1-21 Quality Metrics

Run Quality Metrics

Cluster Density (K/mm²)—Shows the number of clusters per square millimeter

for the Run. For ForenSeq Runs, a target cluster density range of 400–1650 K/mm²

is recommended. Cluster density values outside of the target range can still

produce results that are sufficient to use for analysis. Values that deviate

substantially from the target range can negatively impact other quality metrics

and decrease the quantity of valuable data from the Run.

Clusters Passing Filter (%) – Shows the percentage of clusters passing filter based

on the Illumina chastity filter, which measures quality. The filter can detect low

quality base calls. Data appear only after cycle 25.



NOTE

The chastity of a base call is the ratio of the intensity of the greatest signal divided by the

sum of the two greatest signals. If more than one base call has a chastity value of less than

0.6 in the first 25 cycles, reads do not pass the quality filter.

NOTE

For ForenSeq Samples, a cluster passing filter target value of ≥ 80% is recommended.

Clusters passing filter values that are outside of the target range can still produce results

that are sufficient to use for analysis. Values that deviate substantially from the target

range can negatively impact other quality metrics and decrease the quantity of data from

the Run.

Phasing (%)— Shows the percentage of molecules in a cluster that fall behind the

current cycle within Read 1 and Read 2. Lower percentages are indicative of

higher quality Run statistics. For ForenSeq Samples, a phasing value of ≤ 0.25% is

recommended. Phasing values outside of the target range can still produce results

that are sufficient to use for analysis.

Prephasing (%) – Shows the percentage of molecules in a cluster that Run ahead

of the current cycle within Read 1 and Read 2. Lower percentages are indicative of

higher quality Run statistics. For ForenSeq Samples, a prephasing value of ≤ 0.15%

is recommended. Prephasing values outside of the target range can still produce

results that are sufficient to use for analysis.

Table 4 Run Quality Metrics Color Indicators

Color Indicator

Indicates that values are within the target range.

Indicates the possibility that further evaluation of the quality of the

Run may be beneficial.

Indicates the target or preferred range.

Reads and Index Quality Metrics: ForenSeq mtDNA Control Region Kit

Read 1—Read 1 follows the Read 1 sequencing protocol using the MiSeq FGx

Reagent Kit or MiSeq FGx Reagent Micro Kit. The Read 1 sequencing primer is

annealed to the template strand during the cluster generation step. The RTA

software evaluates the first 50 cycles of the Read 1 segment of the Run for quality.

Read 1 quality metrics are displayed only after cycle 151 is complete.

Index 1—The Read 1 product is removed, and the Index 1 (i7) sequencing primer

is annealed to the same template strand as in Read 1. Following Index Read

preparation, the Index 1 (i7) Read performs eight cycles of sequencing. The RTA

software evaluates all eight cycles of the Index 1 segment of the Run for quality.

Index 1 quality metrics are displayed only after cycle 159 is complete.

Index 2—The Index 1 (i7) Read product is removed, and the template anneals to

the grafted P5 primer on the surface of the MiSeq FGx flow cell. The Run proceeds

through an additional seven chemistry-only cycles in which no imaging occurs,

followed by eight cycles of sequencing. The RTA software evaluates all eight



cycles of the Index 2 (i5) segment of the Run for quality. Index 2 quality metrics

are displayed only after cycle 167 is complete.

Read 2—The Index Read 2 product is extended to copy the original template

strand. Then, the original template strand is removed and the Read 2 sequencing

primer is annealed. Read 2 continues for 30 cycles to sequence through the reverse

PCR primer SBS reagents. The RTA software evaluates all cycles of the Read 2

segment of the Run for quality. Read 2 quality metrics are displayed only after

cycle 318 is complete.

Table 5 Cycle Numbers and Sequencing Phases: ForenSeq mtDNA Control Region Kit

Cycle Number Sequencing Phase

1 to 151 Read 1

152 to 159 Index 1

160 to 167 Index 2

168 to 318 Read 2

Table 6 Read and Index Metrics Color Indicators

Color Indicator

Indicates that the average quality for assessed reads is within the

target range.

Indicates that the average quality for assessed reads is not within the

target range.

Indicates that the read or index did not occur yet in the sequencing

Run.

Table 7 Read and Index Metrics Icons

Icon Indicator

Indicates that the average quality, for Reads 1 and 2 and for Index 1

and 2, are not within the target range.

Indicates that the average quality, for Reads 1 and 2 and for Index 1

and 2, are not within the target range.

Indicates that the Read or Index is pending.

Human Sequencing Control

The loci in the ForenSeq Human Sequencing Control are assessed for an expected minimum

intensity level and the correct genotype. If the overall intensity status fails, then a list of

relevant loci is displayed.



Table 8 ForenSeq Human Sequencing Control Color Indicators

Color Indicator

Indicates that the Sample meets or exceeds minimum intensity criteria.

Indicates that the Samples do not meet one or more criteria. If the bar

is orange, then it is possible that the overall intensity for HSC is lower

than expected. If any loci are listed on the page, then those loci do not

meet intensity or genotype concordance criteria, and their locus name

is listed. For a complete list of HSC loci, see Human Sequencing

Control Loci.

Sample Representation

Sample Representation for the Run shows an overall view of the number of reads and read

distribution for each Sample.

To view the coverage for each Sample, select the Sample Representation indicator

in Run Details.

A dialogue box appears displaying a bar graph that shows the number of reads

for each Sample and the read distribution for each Sample in the Run.

Mouse over the bar to view the exact number of reads for a Sample. Samples are

listed and analyzed with both color and numeric indicators.

Figure 1-22 Sample Representation



Index CV

Displayed as a percentage, the number of reads that are assigned to each Sample. CV is the

coefficient of variation for the number of read counts across all indexes. Index CV represents

the distribution of read counts of the Samples in the Run.

Table 9 Sample Representation Color Indicators

Color Indicator

Indicates a Sample that meets or exceeds the general read count

guideline of 50,000 reads.

Indicates the Sample Representation guideline range (0 - 50,000 reads).

Interpret Samples below 50,000 reads with caution, as Samples with

lower coverage may or may not supply enough reads for variants to

make a call.

Indicates a Sample that has fewer total reads than the general read

count guideline . This is not a hard cut-off. Samples with fewer than

50,000 total reads may or may not include enough data for

interpretation.

Sample Representation Read Count Guideline

A Sample Representation read count guideline of 50,000 reads (per Sample) is supplied by the

software. As described below, complete coverage and calls at 100% of positions of the

mtDNA control region may be observed from Samples with less than 50,000 reads. Thus, this

is a general guideline (not a hard cut-off) that may be helpful in quality review at the Run and

Sample levels.

Data were generated on the MiSeq FGx instrument using the ForenSeq mtDNA Control

Region Kit. Data from two MiSeq FGx Runs (using the MiSeq FGx Reagent Micro Kit) were

analyzed, using the “Verogen mtDNA Control Region Analysis Method” (64 Minimum Read

Count, 10% Analytical and Interpretation Thresholds) as follows:

Sensitivity Study: A dilution series of ForenSeq mtDNA Positive Amplification

Control Samples were Run in quadruplicate (HL-60 @ 1, 2, 5, 10, 20, 50, 100, 200,

500, 1000, 10,000pg input) and a Negative Amplification Control (no template;

water only) was Run in triplicate for a total of 47 Samples.

Total reads per Sample for the 44 HL-60 Samples in the dilution series ranged

from 25,272 to 77,132. Complete coverage of all positions was observed, and all

variants were accurately called, even for Samples with counts <50,000 (e.g. 1pg

gDNA with 25,272 reads).

Known Samples: 46 high quality gDNA Samples (Coriell Institute, Camden, NJ), a

2800M Sample (Promega, Madison, WI) and the ForenSeq mtDNA Positive

Amplification Control (HL-60) at 100pg input each were Run for a total of 48

Samples.



Total reads for 10 Samples were greater than 50,000; complete coverage of all

positions was observed for these Samples. Less than 50,000 total reads were

observed for 38 Samples; complete coverage was observed for 26 of these

Samples. Observations of the remaining 12 Samples were as follows: a range of

1-5 positions in the HVII C-stretch were not called in seven Samples; ~50 positions

were not called in five Samples.



Sample and Run Results

Introduction ........................................................................................................... 38 Viewing a Project .................................................................................................. 39

Navigating to a Project ..................................................................................... 39 Project Summary ............................................................................................. 39 Editing A Project .............................................................................................. 40 Expanding and Collapsing Run Data ............................................................... 40 Sample Summary Searching ........................................................................... 41 Sample Summary Filtering .............................................................................. 42

Viewing Quality and Control Metrics ..................................................................... 43 Quality and Control Metrics ............................................................................. 43 ForenSeq mtDNA Control Region Positive Control Metrics ............................ 43 Negative Control Metrics ................................................................................. 46 Quality Metrics ................................................................................................. 47 Sample Representation ................................................................................... 47

Viewing Sample Details ........................................................................................ 48 Sample Details ................................................................................................. 48 Sample Details Header .................................................................................... 49

Path to Analysis Folder ............................................................................... 49 Selecting an Analysis Method .................................................................... 50

mtDNA Navigator ............................................................................................. 51 Coverage Plot .................................................................................................. 51 Position Viewer ................................................................................................ 52 Filtering Sample Details ................................................................................... 53

Filters and Sample Types ........................................................................... 53 Selecting Filters .......................................................................................... 54

Setting Max Coverage in Sample Details ........................................................ 54 Modifying Results ............................................................................................ 55

Adding Samples to a Project................................................................................. 57 Adding Samples ............................................................................................... 57

Reanalyzing Samples ........................................................................................... 58 Reanalyzing Samples in a Project ................................................................... 58



Introduction

Projects provide the post-sequencing workspace. In Projects, information pertaining to Sample

details are accessible. Quality Metrics, Sample Representation, as well as Positive and

Negative control results, are designed as the launch point for further analysis. Sample details

can be viewed, modified and reanalyzed.



Viewing a Project

When Samples are added to a Run, each Sample is associated to a Project. Each Sample can be

independently assigned to one or more Projects. Information and results for a Project are

viewed in a series of pages in the software interface from the Project page.

Navigating to a Project

There are several ways to locate a Project:

Access the Project from the Run Details Page of your completed Run.

Access the Project from a list of Projects in the system dashboard.

On the system dashboard, use page navigation at the bottom of the Project list to

locate the Project.

On the system dashboard, search by Project Name or Project Description.

Project Summary

Once a Project has been selected, the Project Summary appears. The Project Summary displays

a Project sidebar which lists all Samples grouped by Run. Quality Metrics are retrievable by

hovering over the PNQ indicators in the Project sidebar.

The Project sidebar is further organized into a Samples tab and a Reports tab. The Samples tab

contains a Sample Summary that displays Sample Name, Run Name, Analysis Method,

Sample Type and Assay Type for each Sample analysis. Specific Sample data is accessible

through the Sample link from both the Project sidebar and the Sample Summary. The Reports

tab allows for the creation of Reports and maintains a list of generated Reports.

Figure 2-1 Project Summary Page



Editing A Project

1. Select the pencil icon next to the Project Name to start edit mode.

A dialog box appears.


3. Select Save.

Figure 2-2 Editing a Project

Expanding and Collapsing Run Data

The Project contains a Run and Sample hierarchy listed in the Project sidebar. The Run data

can be collapsed (or expanded) by selecting the arrowhead icon next to the Run Name. Once

selected, associated Samples are no longer visible from below the selected Run Name.

Figure 2-3 Expanded Run Data



Figure 2-4 Collapsed Run Data

Sample Summary Searching

Sample data is retrievable using the Search tool in the Sample Summary.

1. Click inside the Search Bar.

2. Enter a Sample Name, Run Name or Analysis Method.

The Search Bar returns results that meet the search criteria.



Figure 2-5 Sample Summary Search

Sample Summary Filtering

The Filter tool sorts data according to specific parameters.

1. Select the filter icon in either the Sample Type or Assay Type columns.

2. Select an option from the list by selecting a specific box.

3. Select the filter icon.

The filter returns results as necessary.

Figure 2-6 Sample Type Filters in Sample Summary

Figure 2-7 Assay Type Filters in Sample Summary



Viewing Quality and Control Metrics

Quality and Control Metrics

The Project sidebar contains a link to the Quality Metrics, Sample Representation metrics,

Positive Control metrics, and Negative Control metrics, grouped by Run. These tools facilitate

data analysis for each Sample (in the context of the associated Run), allowing for both a

general and specific perspective on the data.

Figure 2-8 Quality and Control Metrics

ForenSeq mtDNA Control Region Positive Control Metrics

The Positive Control tab lists Samples identified as Positive Amplification Control. Each

Sample is analyzed across the amplified mtDNA coordinates and compared with the known

ForenSeq Positive Control DNA (HL-60) multi-locus genotype. Positive amplification controls

are displayed with both color and numeric indicators.

From the Positive Control tab, the following information is displayed for each positive

amplification control Sample:

The name of the positive amplification control Sample, which can be selected to

open the Sample Details page.

The number of positions for which a base call was made out of the total number of

positions for the mtDNA control region of ForenSeq Positive Control HL-60

A list of discordant positions compared to ForenSeq Positive Control HL-60, if

applicable.

Total number of reads



Figure 2-9 ForenSeq mtDNA Control Region Positive Control Metrics (HL-60)

Next to each positive amplification control Sample is a short vertical color bar indicating the

number of positions with a call out of the total number of positions.

Table 10 ForenSeq mtDNA Positive Amplification Control Quality Color Indicators

Color Indicator

Indicates that all positions contain calls and are concordant with the

known ForenSeq mtDNA Positive Control DNA HL-60.

Indicates that at least one position does not contain a call or contains

a call that is discordant with the known ForenSeq mtDNA Positive

Control DNA HL-60.

If warranted, a discordance table shows discordant positions compared to ForenSeq mtDNA

Control HL-60 and includes the following information:

Position of discordance

Observed call in the Sample

Expected ForenSeq mtDNA HL-60 call



Figure 2-10 Discordance for a ForenSeq mtDNA Positive Amplification Control

NOTE

Positions in the Discordance box are viewable by scrolling along the right-hand

column. When more than 50 positions are discordant, navigate to the next page

using the arrows at the bottom of the box.

Table 11 ForenSeq mtDNA Positive Amplification Control Pass Filter Reads Color Indicators

Color Indicator

Indicates that the total number of reads is greater than 50,000

Indicates that the total number of reads is less than 50,000. It is

possible to use these data, however review of the quality results on the

Quality Metrics page and data on Sample Details page may be

beneficial.

Indicates a guideline of 50,000 reads or more.



Negative Control Metrics

The Negative Control tab lists Samples identified as Negative Amplification Control or

Reagent Blank. Each Sample is analyzed across the amplified ForenSeq mtDNA coordinates.

Negative Amplification Controls are displayed with both color and numeric indicators.

From the Negative Control tab, the following information is displayed for each Negative

Amplification Control or Reagent Blank:

The Sample Name of the Negative Amplification Control , which can be selected

to open the Sample Details page.

The Sample Name of the Reagent Blank, which can be selected to open the Sample

Details page.

The number of positions for which a base call was made.

Figure 2-11 Negative Control Metrics

Next to each Negative Amplification Control or Reagent Blank is a short vertical color bar

indicating the number of mtDNA positions with a call out of the total number of positions in

the rCRS.

Table 12 Negative Amplification Control Quality Color Indicators for mtDNA

Color Indicator

Indicates that no positions contain a call.

Indicates that at least one position contains a call.



Quality Metrics

Figure 2-12 Quality Metrics in Projects

For an explanation on Quality Metrics, please refer to Quality Metrics.

Sample Representation

Figure 2-13 Sample Representation in Projects

For an explanation on Sample Representation, please refer to Sample Representation.



Viewing Sample Details

Sample Details

When a Sample is selected from the Project summary, the Sample Details page is displayed.

The Sample Details page is the workspace for in-depth data analysis. Comprised of the

mtDNA Navigator (A), the Position Viewer (B), and the Coverage Plot (C), the Sample Details

page facilitates examination of mtDNA sequence data. Whether moving the pointer around

the mtDNA control region, specifying a coverage threshold, or searching for variants, the tools

work in unison expediting the analytical process. Examination of positions, variants, coverage,

interpretation thresholds, user modifications, or the interplay amongst these attributes is

accomplished on a single page with each tool providing a specific function based upon user

needs.

The Sample Details page contains the following information:

Sample Name

Sample Description (if an optional description was included)

Analysis Folder Path

Analysis Method

Analysis State

Sample Type

Date and time the Sample analysis was created in the ForenSeq Universal

Analysis Software

Date and time the Sample analysis was completed in the ForenSeq Universal

Analysis Software

Filter function

Sample Compare function

Create Report function

mtDNA Navigator

Position Viewer

Coverage Plot



Figure 2-14 Sample Details

A. Sample Details Header

B. mtDNA Navigator

C. Coverage Plot

D. Position Viewer

Sample Details Header

The Sample Details Header contains the following information:

Sample Name

Sample Description (if an optional description was included)

Analysis Folder Path

Analysis Method

Analysis State

Sample Type

Date and time the Sample analysis was created in the ForenSeq Universal

Analysis Software

Date and time the Sample analysis was completed in the ForenSeq Universal

Analysis Software

Path to Analysis Folder

The Analysis Folder Path can be accessed by selecting the blue information icon next to

Sample Name. Selecting the icon displays a dialogue box containing the path to the analysis

files from the ForenSeq Analysis Software server.

C

B D

A



Figure 2-15 Analysis Folder Path

Selecting an Analysis Method

The Sample Details page displays the name of the Analysis Method used to generate the

results displayed in the mtDNA Navigator, Coverage Plot and Position Viewer. Each Sample

will be associated with the Run Analysis Method, at minimum, and one more additional

analysis if a user re-analyzes with a different Analysis Method.

Figure 2-16 Analysis Method

If a Sample was reanalyzed, the Analysis Method dropdown will list all Analysis Methods

used to generate results, with the most recent Analysis Method appearing first. Selecting an

Analysis Method from the dropdown will update the mtDNA Navigator, Coverage Plot and

Position Viewer with results that correspond to the selected Analysis Method.



Figure 2-17 Multiple Analysis Methods

mtDNA Navigator

The mtDNA Navigator default view displays sequencing results that span the control region

in the zoomed view.

Figure 2-18 mtDNA Navigator

A. Control Region

B. Max Coverage

C. Pointer

Coverage Plot

The Coverage Plot displays the mtDNA Position (x-axis) and Coverage (number of reads, y-

axis). The selected position is marked by the black cursor, which can be moved causing a

simultaneous shift within the Position Viewer and the mtDNA Navigator pointers (views

above the Coverage Plot).

A

B

C



Figure 2-19 Coverage Plot, no Max Coverage

Position Viewer

The Position Viewer displays the selected Position, rCRS nucleotides and sequenced Sample(s)

nucleotide calls. Inputting a different position number automatically aligns the rCRS and

Sample call accordingly, as well as the mtDNA Navigator pointer and the Coverage Plot

pointer.

Figure 2-20 Position Viewer

A. Previous hotspot

B. Previous page

C. Position Number

D. Next hotspot

E. Next page

F. rCRS Call

G. Sample Call

NOTE

Move across the Position Viewer by using the up and down arrows keys on the

keyboard or by selecting a position with a cursor.

A

B

C

D

E

Pointer

F

G



Filtering Sample Details

Prior to engaging with tools on the Sample Details page, the entire circular mtGenome is

shown as the default view in the mtDNA Navigator and the Coverage Plot. The software

allows for closer examination of the data through the Filter button and Max Coverage bar.

Filters and numeric inputs into the Max Coverage bar affect the mtDNA Navigator, the

Position Viewer and the Coverage Plot simultaneously. Clicking directly on a position will

move all three tools simultaneously as well.

The Filter dropdown contains the following filter options:

Control Region

Interpretation Threshold

Variants

No Call (for Samples and Positive Amplification Controls)

Call (for Reagent Blanks and Negative Amplification Controls)

Filters and Sample Types

The No Call and Call filters exhibit specific behaviors and differ from the Control Region,

Interpretation Threshold and Variants filters. The No Call and Call filters are dependent on

Sample Type.

When the Sample Type is the ForenSeq Positive Amplification Control or Sample, the No Call

filter will display in the filter dropdown. Additionally, the mtDNA Navigator and Position

Viewer will indicate positions with no calls.

Figure 2-21 Filters for ForenSeq Positive Amplification Controls and Samples



When the Sample Type is a Negative Amplification Control or Reagent Blank, the Call filter

will display in the filter dropdown. Additionally, the mtDNA Navigator and Position Viewer

will indicate positions with calls.

Figure 2-22 Filters for Negative Amplification Controls and Reagent Blanks

Selecting Filters

1. Select the filter icon to view a list of filter options.

2. Select one or more filters from the list by selecting a specific box.

The filter icon changes to indicate the total number of active filters.

3. Select the filter icon to return results as necessary.

The mtDNA Navigator, Coverage Plot and Position Viewer will update

simultaneously when a filter is selected, as applicable.

Setting Max Coverage in Sample Details

The Max Coverage bar enables filtering by total read count. Selecting a max coverage value

updates the mtDNA Navigator and the Coverage Plot simultaneously, and acts as a zoom

function.

1. Enter a read number in the Max Coverage bar.

NOTE

Input read numbers without commas.

2. The mtDNA Navigator and the Coverage Plot (y-axis ) adjust to the set Max

Coverage.



Figure 2-23 Setting Max Coverage

Modifying Results

Sample calls for each position can be modified in the Position Viewer.

1. In the Position Viewer, navigate to a position requiring modification.

In the Call column, a dropdown menu displays a list of available calls. The calls are

grouped by Unambiguous and Ambiguous calls.

2. To change a call, make a selection from the dropdown menu.

The Position Viewer is updated to reflect the modified call and a User-Modified

Indicator is added at the modified position. Additionally, the original call is added in

the Call column beneath the dropdown menu.

3. To change the call back to the original call, make the appropriate selection from the

dropdown menu.

The User-Modified Indicator is removed from the position.



Figure 2-24 Modifying Results



Adding Samples to a Project

Adding Samples

Analyzed Samples that reside within a Project can be added to other Projects. A Sample must

reside in a Project before it can be added to another Project. When a Sample is added to

another Project, any user modifications to the Sample will not be carried over into the newly

assigned Project.

To add Samples to a Project:

1. From the Project page, select Add Sample(s).

A dialog box appears.

3. Enter a Sample Name, Run Name, or Project Name.

The dialog box populates with results based upon entered search criteria.

4. Select the Samples to be added by selecting the box next to the Sample Name.

The Summary column displays all Sample selections with corresponding analysis

methods.

5. Select Save.

NOTE

To deselect a Sample, select the X next to the Sample Name in the Summary

column.

Figure 2-25 Adding Samples to a Project



Reanalyzing Samples

Reanalyzing Samples in a Project

Samples can be reanalyzed within Projects. Reanalysis allows for the selection of Analysis

Methods with user-defined parameters, as desired.

To reanalyze Samples:

1. From the Sample Summary page, select Sample(s) for reanalysis by selecting the box

next to the Sample Name.

2. Select the Reanalyze button.

A dialog box appears listing all of the selected Sample(s).

NOTE

Samples selected for reanalysis are grouped by Assay Type. The Analysis Method

dropdown only lists Analysis Methods per Assay Type.

3. Select a new Analysis Method from the dropdown.

One Sample: To assign an Analysis Method to an individual Sample, select the

method from the box to the right of the Sample Name.

Multiple Samples: To assign an Analysis Method to all listed Samples, select the

method from the box above the Sample Name column, and select Apply to All.

Figure 2-26 Select Sample for Reanalysis

4. Select Next.

5. Review the Sample Name, Previous Analysis Method, and the selected New Analysis

Method columns.

If there is a correctable discrepancy, select Previous to return to the original dialog

box and make the necessary changes. Proceed as above.

If there is an uncorrectable discrepancy, select Cancel.



6. Select Save.

Figure 2-27 Review Sample Selected for Reanalysis

7. Upon saving the Sample selections, reanalysis is initiated. The Project Summary

indicates Samples undergoing reanalysis with an icon to the right of the Sample

Name.

The Sample Details page indicates reanalysis by displaying the Analysis State as

Processing. Once reanalysis is complete, results will display on the Sample Details

page.

Figure 2-28 Sample Reanalysis



Sample Comparison

Introduction ........................................................................................................... 61 Sample Comparison ............................................................................................. 62

Comparing Two Samples ................................................................................ 62 Comparing More Than Two Samples .............................................................. 63 Filtering with Sample Compare........................................................................ 64

Selecting Filters During a Sample Compare .............................................. 66 Stopping Sample Compare.............................................................................. 66



Introduction

The ForenSeq Universal Analysis Software Sample comparison feature evaluates results

between two Samples using as many or as few of the filter tools as necessary. Sample

Comparisons are enabled for Samples from the same Project. During a Sample Comparison,

calls may not be modified.



Sample Comparison

Comparing Two Samples

Sample comparisons are initiated from the Sample Details page.

1. Select a Sample from the Project.

In the Sample comparison, this Sample is called the Sample of Interest and will be

indicated in black text when the comparison is initiated.

2. Select the Compare button above the mtDNA Navigator.

A Sample Compare dialogue box appears with a list of all Samples from the Project.

Use the search bar or scroll through the list of Samples to find the second Sample you

want to compare. In the Sample comparison, this Sample is called the Selected

Comparison Sample and will be indicated in blue text when the comparison is

indicated.

Figure 3-1 Sample Selection for Sample Comparison

3. Select a Comparison Sample by selecting the white box next to the Sample Name.

4. Select Submit to initiate the Sample Comparison.

The Sample Details page automatically adjusts to include the Selected Comparison

Sample in the mtDNA Navigator, Position Viewer and Coverage Plot.

The compare icon changes to enable stopping of the comparison when desired.



Figure 3-2 Comparing Two Samples

NOTE

The Sample of Interest is colored gray in the mtDNA Navigator and the Coverage

Plot, while the comparison Selected Comparison Sample is colored blue. In the

Position Viewer, the highlighted blue row designates the Selected Comparison

Sample, while the Sample of Interest remains in black text.

Comparing More Than Two Samples

1. Select a Sample from the Project.

In the Sample comparison, this Sample is called the Sample of Interest and will be

indicated in black text when the comparison is initiated.

2. Select the Compare button above the mtDNA Navigator.

A Sample Compare dialogue box appears with a list of all Samples from the Project.

Use the search bar or scroll through the list of Samples to find the second Sample you

want to compare. In the Sample comparison, these Samples are called the Selected

Comparison Samples and will be indicated in blue text when the comparison is

indicated.

NOTE

A maximum of eight Samples can be selected for comparison.

3. Select Comparison Samples by selecting the white box next to the Sample Names.

4. Select Submit to initiate the Sample comparison.

The Sample Details page automatically adjusts to include the selected Sample in the

mtDNA Navigator, Position Viewer and Coverage Plot.

The compare icon changes to enable stopping of the comparison when desired.



5. To alternate between Selected Comparison Samples, select the desired Sample Name

in blue text.

Figure 3-3 Comparing More Than Two Samples

NOTE

The Sample of Interest is colored gray in the mtDNA Navigator and the Coverage

Plot, while the comparison Selected Comparison Samples are colored blue. In the

Position Viewer, the highlighted blue row designates the Selected Comparison

Samples, while the Sample of Interest remains in black text.

Filtering with Sample Compare

Filtering enables data interpretation of flagged calls in the Sample of Interest and Selected

Comparison Sample(s).

The Filter dropdown during a Sample Compare contains the following filter options:

Control Region


Variants

Differences

No Call (ForenSeq Positive Amplification Controls and Samples)

Call (Negative Amplification Controls and Reagent Blanks)



The Differences filter highlights call differences between the Sample of Interest and the

Selected Comparison Sample(s).

The No Call and Call filters exhibit specific behaviors and differ from the Control Region,

Interpretation Threshold, Variants and Differences filters. The No Call and Call filters are

dependent on the Sample Type of the Sample of Interest.

When the Sample of Interest is the ForenSeq Positive Amplification Control or

Sample, the No Call filter displays in the filter dropdown. Additionally, the

mtDNA Navigator and Position Viewer will indicate positions with no calls.

Figure 3-4 Filtering a Sample Comparison with a ForenSeq Positive Amplification Control or Sample

NOTE

In a Sample Comparison, when a ForenSeq Positive Amplification Control or

Sample is designated as the Sample of Interest, the No Call filter is enabled, and

the Call filter is not.

When the Sample of Interest is a Negative Amplification Control or Reagent

Blank, the Call filter displays in the filter dropdown. Additionally, the mtDNA

Navigator and Position Viewer will indicate positions with calls.



Figure 3-5 Filtering a Sample Comparison with a Negative Amplification Control or Reagent Blank

NOTE

In a Sample Comparison, when a Negative Amplification Control or Reagent

Blank is designated as the Sample of Interest, the Call filter is enabled, and the No

Call filter is not.

Selecting Filters During a Sample Compare

1. Select the filter icon to view a list of filter options.

2. Select one or more filters from the list by selecting a specific box.

The filter icon changes to indicate the total number of active filters.

3. Select the filter icon to return results as necessary.

The mtDNA Navigator, Coverage Plot and Position Viewer will update

simultaneously when a filter is selected, as applicable.

Stopping Sample Compare

Sample comparisons can be stopped by selecting Stop Compare. The Sample Details page

returns to the Sample Details page of the Sample of Interest.



Reports

Introduction ........................................................................................................... 68 Project Report ....................................................................................................... 69

Creating a Project Report ................................................................................ 69 Reading a Project Report ................................................................................ 70

Sample Report ...................................................................................................... 72 Creating a Sample Report from the Project Summary .................................... 72 Creating a Sample Report from the Sample Details Page .............................. 73 Reading a Sample Report ............................................................................... 73

CODIS Report ....................................................................................................... 75 Creating a CODIS Report from the Project Summary ..................................... 75 Creating a CODIS Report from the Sample Details Page ............................... 77 Reading a CODIS Report ................................................................................ 79

EMPOP Report ..................................................................................................... 81 Creating an EMPOP Report from the Project Summary ................................. 81 Creating an EMPOP Report from the Sample Details Page ........................... 82 Reading an EMPOP Report............................................................................. 82



Introduction

The ForenSeq Universal Analysis Software features the ability to create and view the

following reports:

Project Report

Sample Report

CODIS Report

EMPOP Report

Reports are created in Projects and can be initiated from either the Sample Details page or

from the Reports tab in the Project sidebar.

From the Reports tab, Reports Summary displays and contains the following information:

A Create Report button enabling report creation.

A search tool enabling searching by file name.

A list of created reports arranged by creation date with the most recent report

appearing at the top of the list.

A filter tool enabling filtering by Report Type.

A download button enabling download of created reports.



Project Report

The Project Report is a downloadable file consisting of one summary worksheet and

additional worksheets for each Sample analysis result associated with the Project.

Creating a Project Report

To create a Project Report, navigate to the Project Summary.

1. Select Reports from the Project Summary.

This action opens the Report Summary.

Figure 4-1 Report Summary

2. Select Create Report from the Report Summary.

A list of reports is displayed.

3. Select Project.

A dialogue box appears requiring confirmation.



Figure 4-2 Create Project Report

NOTE

When one or more Samples are being analyzed, Project Report creation is

disabled. A dialogue box is displayed with the following message: “One or more

Samples are being analyzed. Report creation is available after analyses of

associated Sample(s) are complete.

4. Select Yes.

A Project Report is automatically created and displayed in the Report Summary with

a listing of the Date of Creation, Report Type and File Name.

5. To download the Project Report, select the download icon under the Actions column.

Figure 4-3 Download Project Report

Reading a Project Report

The first worksheet in the Project Report, titled “Variant Summary Report” contains the

following information:

Project Name

Description

Run Count

Sample Count

Project Created Date



List of Samples

Single Nucleotides Variants count per Sample

Insertion count per Sample

Deletion count per Sample

No Call count per Sample

List of manually changed calls per Sample

List of variants per Sample

The subsequent worksheets in the Project Report contain Sample information as delineated in

section Reading A Sample Report.

Figure 4-4 Project Report



Sample Report

The Sample Report is a downloadable file consisting of one worksheet which outlines Sample

details information. Sample Reports can be created from the Reports Tab in the Project

Summary or from the Sample Details page.

Creating a Sample Report from the Project Summary

To create a Sample Report from the Project Summary:





3. Select Sample.

A Create Sample Report dialogue box appears with a list of all Samples from the

Project. Use the search bar or scroll through the list of Samples to find the Sample(s)

you wish to select.

NOTE

When more than one Sample is selected, individual Sample Reports will be

created for each Sample.

Figure 4-5 Create Sample Report

4. Select Confirm.

A Sample Report is automatically created and displayed in the Report Summary with


5. To download the Sample Report, select the download icon under the Actions column.



Figure 4-6 Download Sample Report

Creating a Sample Report from the Sample Details Page

To create a Sample Report from the Sample Details page:

1. Select a Sample from the Project Summary.

This action opens the Sample Details page for the selected Sample.

2. Select Create Report from the Sample Details page.


Figure 4-7 Selecting Sample Report

3. Select Sample.

A Sample Report is automatically created and can be viewed in the Report Summary.

4. To download the Sample Report, select the download icon under the Actions column

in the Report Summary.

Reading a Sample Report

The Sample Report contains the following information:

Sample Name

Assay Type

Analysis Method

Run Name



Analytical Threshold


Minimum Q-Score

Minimum Read Count

Single Nucleotide Variants

Insertion count

Deletion count

No Call count per Sample

List of manually changed calls per Sample

Variant Details

Variant Details is further broken down into the following information for each listed variant:

Variant Call

Reference Call

Total Read Count

Strand Depth

Reads A, C, T, G

Percent A, C, T, G

Reads Del

Percent Del

Reads Ref

Percent Ref

Figure 4-8 Sample Report



CODIS Report

The CODIS Report is a downloadable .cmf file. CODIS Reports can be created from the

Reports Tab in the Project Summary or from the Sample Details page.

Creating a CODIS Report from the Project Summary

To create a CODIS Report from the Project Summary:





3. Select CODIS.

A Create CODIS Report dialogue box appears with a list of all Samples from the

Project. Use the search bar or scroll through the list of Samples to find the Sample(s)

you wish to select.

NOTE

When one or more Samples are selected, a single CODIS Report will be created

for all selected Samples.

Figure 4-9 Create CODIS Report

4. Once Samples have been selected, select Next.

5. Enter the following information for the CMF Header:

Destination Laboratory ORI

Source Laboratory ORI

Submission User ID

NOTE




Figure 4-10 CODIS Report CMF Header

6. Select Next.

7. Select a Specimen Category for each Sample.

A Specimen Category can be selected for each individual Sample or one Specimen

Category can be applied to all Samples.

Figure 4-11 CODIS Report Specimen Categories

NOTE

When one Sample is selected, an additional step for defining Target Information

is displayed.

8. Select Create.

A CODIS Report is automatically created and displayed in the Report Summary with




9. To download the CODIS Report, select the download icon under the Actions column.

Figure 4-12 Download CODIS Report

Creating a CODIS Report from the Sample Details Page

To create a CODIS Report from the Sample Details page:




A list of options for Report types is displayed.

Figure 4-13 Selecting CODIS Report

3. Select CODIS.

A Create CODIS Report dialogue box appears.



Figure 4-14 Selecting CODIS Report CMF Header

4. Enter the following information for the CMF Header:



Submission User ID

NOTE


5. Select Next.

6. Enter the following Specimen Information:

Specimen Category

Source ID

NCIC Number

ViCAP Number

Specimen Comment



Figure 4-15 Selecting CODIS Specimen Information

7. Select Next.

Target Information is displayed with a list of positions for the Sample, along with an

option to deselect positions. Deselected positions will not be included in the CODIS

Report.

8. Deselect desired positions.

9. Select Create.

A CODIS Report is automatically created and displayed in the Report Summary with


10. To download the CODIS Report, select the download icon under the Actions column


Reading a CODIS Report

The CODIS Report contains the following information:

CMF Header

Reference Sequence

Specimen Profile

The Specimen Profile is further broken down into the following information for each listed

Sample:

Specimen ID

Specimen Category

Source ID

Fragments



Figure 4-16 CODIS Report



EMPOP Report

The EMPOP Report is a downloadable .txt file consisting of two formats: the FASTA format

and the rCRS format, both of which are included in the Report. EMPOP Reports can be created

from the Reports Tab in the Project Summary or from the Sample Details page.

Creating an EMPOP Report from the Project Summary

To create an EMPOP Report from the Project Summary:





3. Select EMPOP.

A Create EMPOP Report dialogue box appears with a list of Samples from the Project.

Use the search bar or scroll through the list of Samples to find the Sample(s) you wish

to select.

NOTE

When one or more Samples are selected, a single EMPOP Report will be created

for all selected Samples.

Figure 4-17 Create EMPOP Report

4. Select Confirm.

An EMPOP Report is automatically created and displayed in the Report Summary

with a listing of the Date of Creation, Report Type and File Name.

5. To download the EMPOP Report, select the download icon under the Actions column.



Figure 4-18 Download EMPOP Report

Creating an EMPOP Report from the Sample Details Page

To create an EMPOP Report from the Sample Details page:





3. Select EMPOP.

An EMPOP Report is automatically created and can be viewed in the Report

Summary with a listing of the Date of Creation, Report Type and File Name.

4. To download the EMPOP Report, select the download icon under the Actions column


Reading an EMPOP Report

The EMPOP Report contains the following information:

Project Name

Sample Name

Assay Type

Analysis Method

Run Name

FASTA Format contain a sequence string

rCRS Format contain list of variant calls



Figure 4-19 EMPOP Report



System Settings

Introduction ........................................................................................................... 85 Analysis Methods .................................................................................................. 86

Verogen mtDNA Control Region Analysis Method .......................................... 86 Verogen mtDNA Custom Analysis Method ...................................................... 88 Creating an Analysis Method for mtDNA Control Region ................................ 90 Creating an Analysis Method for mtDNA Custom ........................................... 91 Changing the Default Analysis Method ........................................................... 95 Deleting an Analysis Method ........................................................................... 95

CODIS Report Defaults ......................................................................................... 97 Defining CODIS Defaults ................................................................................. 97 Enabling and Disabling CODIS Specimen Categories .................................... 97 Creating Custom CODIS Specimen Categories .............................................. 98 Editing Custom CODIS Specimen Categories ................................................ 99 Deleting Custom CODIS Specimen Categories ............................................ 100 Enabling and Disabling Custom CODIS Specimen Categories .................... 100

User Management .............................................................................................. 101 Enabling and Disabling Accounts .................................................................. 101 Assigning Account Access Levels ................................................................. 101 Changing an Account Password ................................................................... 102



Introduction

System Settings are accessed from the sidebar menu in the system dashboard. System Settings

enable maintenance of the following:

Analysis Methods

CODIS Report Defaults

Administrator Settings

Analysis Method settings deal with aspects such as quantitative values and thresholds for

data analysis.

Values specified in CODIS Report Defaults pre-populate in the CODIS Report generator

workflow to expedite Report generation. These default values can be changed at the time of

Report generation, if desired.

Administrator Settings allow for user management by enabling and disabling user access.

Figure 5-1 System Settings



Analysis Methods

An Analysis Method is a collection of settings that inform allele or variant calls from

sequencing data. Each of these settings can be user-adjusted and are a matter of internal

laboratory policy-making.

A default Analysis Method is supplied for the following Assay Types:

ForenSeq mtDNA Control Region

mtDNA Custom

NOTE

Default Analysis Methods cannot be modified. To create a new Analysis Method,

Copy one of the default Analysis Methods listed above. This workflow is detailed

in subsequent sections.

Verogen mtDNA Control Region Analysis Method

The Verogen mtDNA Control Region Analysis Method is the default Analysis Method for the

ForenSeq mtDNA Control Region Assay Type. This Analysis Method is comprised of the

following settings and their corresponding defaults:

Analytical Threshold (AT): A nucleotide with total number of reads that are less than the AT

value do not contribute to a call.

Default Analytical Threshold (AT): Background signal was assessed on four

MiSeq FGx instruments by four operators, using the ForenSeq mtDNA Control

Region Kit. Data were generated in order to determine the percentage of reads for

the default AT that is applied in the Verogen mtDNA Control Region Analysis

Method. Ten MiSeq FGx Runs (using MiSeq FGx Reagent Micro Kits) with a total

of 63 ForenSeq Positive Amplification Controls (100 pg input, HL-60 control

DNA), were evaluated. Total numbers of Samples per Run ranged from two to

18. Data were analyzed using the default 64 Minimum Read Count, with the AT

and Interpretation Threshold (IT) set to 0%.

Unexpected variants observed in the 63 positive control Samples were collated, as

follows, for the 10 Runs:

o Average unexpected variant percentage observed was 0.7% (1% standard

deviation) and ranged from 0.1 – 9.7% (4,157 out of 72,954 nucleotides).

o 95th percentile for unexpected variants was 2.2%.

▪ Range of the maximum percentages of unexpected variants was 3.9 –

9.7%; each max data point occurred within C-stretches of HVI and HVII,

or the AC repeat at positions 523-524.

▪ Excluding these locations, the maximum unexpected variants observed

was 2.1%.

o The default AT was conservatively set to 10% in the Verogen mtDNA Control

Region Analysis Method as follows:



▪ A 10% AT (maximum unobserved variant % rounded to the nearest

whole integer) provides the known HL-60 haplotype from these 63

Positive Control Samples.

▪ If 3.7% is used as AT (0.7% mean +3SD), then 3.5% of unexpected

variants are called (147 of 4,157), all in either the C-stretches or the AC

repeat at positions 523-524.

▪ An AT less than 10% may be desired, depending upon internal lab

needs.

▪ For example, if detection heteroplasmy or contributors in mixtures is

desired at <10%, lowering the AT may be considered. Data described

above indicate that a 3.7% AT can assist with detection and

interpretation of heteroplasmy or minor contributors at 5%, with care

taken in interpreting HVI and HVII C-stretches and the 523-524 AC

repeat.

Interpretation Threshold (IT): A nucleotide with total number of reads that are greater than

or equal to the IT contribute to a call for the coordinate. When more than one base exceeds the

IT for a coordinate, the IUPAC Code for the bases is reported.

Default Interpretation Threshold (IT): Set to 10%, same as the default Analytical

Threshold. The IT is user-adjustable and is a matter of laboratory policy-making.

Minimum Quality Score: Q302 is the default setting for Minimum Quality Score (Q Score).

The Minimum Q Score at a position must be reached in order to be considered for base calling.

Minimum Read Count: 64 nucleotide-specific reads is the default setting for Minimum Read

Count. The Minimum Read Count must be reached at a position for a nucleotide in order to

be considered for base calling.

Default Minimum Read Count: A lower limit (nucleotide-specific read number),

used to assign a nucleotide call in the mitochondrial control region, is supplied to

the ForenSeq Universal Analysis Software (UAS) by the Minimum Read Count

value in an Analysis Method.

Background signal was assessed on two MiSeq FGx instruments using the

ForenSeq mtDNA Control Region Kit. Data were generated in order to determine

the default Minimum Read Count value applied in the Verogen mtDNA Control

Region Analysis Method. Two MiSeq FGx Runs (using a MiSeq FGx Reagent

Micro Kit) of 48 Negative Amplification Controls (no template; water only) were

evaluated. Data indicate an average of 34 reads per position in these NTCs, with a

standard deviation of 30 reads across the 1,157 positions (mtDNA control region)

evaluated. A default value of 64 reads per nucleotide at a position, as calculated

2 Quality Scores for Next-Generation Sequencing. (n.d.). [ebook] San Diego: Illumina, pp.1-2.

Available at: https://www.illumina.com/content/dam/illumina-

marketing/documents/products/technotes/technote_Q-Scores.pdf.



by one standard deviation above the mean read number, is thus the default

Minimum Read Count value applied in the Verogen mtDNA Control Region

Analysis Method.

The Minimum Read Count value is user-adjustable) and is a matter of laboratory

policy-making.

If 64 reads is used as the default Minimum Read Count, then a call and coverage

is not reported for a position with 63 or fewer reads for a specific nucleotide (A, G,

C or T); when 64 or more reads are present at a position for one or more

nucleotides (A, G, C, T), a call (with associated coverage) is reported, if compliant

with other analysis parameters. A call is compliant at a position when the

following are met or exceeded: Minimum Q-Score (Q30), AT% and Minimum

Read Count.

For example, when 64 reads are used as the default Minimum Read Count, and

100 thymine (T) reads are present at a position that meet Q30 and AT%, then a call

is made. When 64 reads are used as the default Minimum Read Count, and 50

thymine (T) reads plus 50 adenine (A) reads are present at a position, then a call is

not made and the coverage is not reported, regardless of whether Q30 and AT%

are met.

Figure 5-2 Verogen mtDNA Control Region Analysis Method

NOTE

The Verogen mtDNA Control Region Analysis Method is a Primer-Directed

Sequencing Library Type. This setting is pre-set in the default Analysis Method

for the ForenSeq mtDNA Control Region Assay Type and is not user-defined.

Verogen mtDNA Custom Analysis Method

A Custom mtDNA Analysis Method is required to analyze mtDNA sequencing data, from the

MiSeq FGx, that are generated from other library preparation methods. Custom mtDNA



Analysis Methods are comprised of the four user-defined settings described above for the

Verogen mtDNA Control Region Analysis Method plus an additional setting, Library Type.

Custom mtDNA data may be generated on the MiSeq FGx from two Library Types: Non-

Directed Sequencing or Primer-Directed Sequencing. An example of Non-Directed Sequencing

is one that uses a transposase-based approach, such as Nextera XT. An example of Primer-

Directed Sequencing is one that uses a PCR-based approach to create sequencing targets

(amplicons), such as TruSeq Custom Amplicon Library Prep.

The Verogen mtDNA Custom Analysis Method is a templated Analysis Method for use with

the mtDNA Custom Assay Type. This Analysis Method is comprised of the following settings

and their corresponding defaults:

Analytical Threshold (AT): 10% is the default setting for Analytical Threshold. A

nucleotide with total number of reads that are less than the AT do not contribute

to a call. The AT can be used to represent a lower limit of detection.

Interpretation Threshold (IT): 25% is the default setting for Interpretation

Threshold. A nucleotide with total number of reads that are greater than or equal

to the IT contribute to a call for the coordinate. When more than one base exceeds

the IT for a coordinate, the IUPAC Code for the bases is reported.

Minimum Quality Score: Q303 is the default setting for Minimum Quality Score (Q

Score). The Minimum Q Score at a base must be reached in order for that

coordinate to be considered for variant calling.

Minimum Read Count: 64 nucleotide-specific reads is the default setting for

Minimum Read Count. The Minimum Read Count must be reached at a position

for a nucleotide in order to be considered for base calling.

Reference Genome: The Revised Cambridge Reference Sequence (rCRS) is the

default Reference Genome.

Library Type: Non-Directed Sequencing is the default Library Type and uses a

library preparation protocol4 that targets the whole mtDNA genome.

The Non-Directed Sequencing Library Type for the Verogen mtDNA Custom

Analysis Method is comprised of the following information:

Amplicon Name

Forward Primer Start Coordinate

Reverse Primer Start Coordinate

Forward Primer Length

3 Quality Scores for Next-Generation Sequencing. (n.d.). [ebook] San Diego: Illumina, pp.1-2.

Available at: https://www.illumina.com/content/dam/illumina-

marketing/documents/products/technotes/technote_Q-Scores.pdf.

4 Human mtDNA Genome. (2013). [ebook] Illumina, p.53. Available at:

https://verogen.com/wp-content/uploads/2018/07/human-mtdna-genome-guide-15037958-

01.pdf



Reverse Primer Length

The Non-Directed Sequencing Library Type for the Verogen mtDNA Custom

Analysis Method contains two amplicons with the following defaults:

Amplicon Name: MTL F1 – MTL R1

Forward Primer Start Coordinate: 9397

Reverse Primer Start Coordinate: 1892

Forward Primer Length: 20

Reverse Primer Length: 20

Amplicon Name: MTL F2 – MTL R2

Forward Primer Start Coordinate: 15195

Reverse Primer Start Coordinate: 9796

Forward Primer Length: 20

Reverse Primer Length: 20

Figure 5-3 Verogen mtDNA Custom Analysis Method

Creating an Analysis Method for mtDNA Control Region

1. Select Settings.

2. Select Analysis Methods.

3. Select ForenSeq mtDNA Control Region from the Assay Type dropdown.

4. Select Verogen mtDNA Control Region Analysis Method from the Analysis Method

dropdown.

5. Select Copy.

A Copy Analysis Method dialogue box appears.

6. Enter the name of the new Analysis Method.



Figure 5-4 Create New mtDNA Control Region Analysis Method

7. Select Save.

A new Analysis Method is created containing default values.

8. To modify the Analytical Threshold and Interpretation Threshold, adjust the

percentages as needed.

9. To modify the Minimum Quality Score and Minimum Read Count, adjust the values

as needed.

10. Select Save.

NOTE

All values selected for Analytical Threshold, Interpretation Threshold, Minimum

Quality Score, and Minimum Read Count are considered “greater than or equal

to.”

Creating an Analysis Method for mtDNA Custom

1. Select Settings.


3. Select mtDNA Custom from the Assay Type dropdown.

4. Select Verogen mtDNA Custom Analysis Method from the Analysis Method

dropdown.

5. Select Copy.

A Copy Analysis Method dialogue box appears.

6. Enter the name of the new Analysis Method.



Figure 5-5 Create New mtDNA Custom Analysis Method

7. Select Save.

A new Analysis Method is created containing the default values.

8. To modify the Analytical Threshold and Interpretation Threshold, adjust the

percentages as needed.

9. To modify the Minimum Quality Score and Minimum Read Count, adjust the values

as needed.

10. To remove the listed amplicon(s) for Non-Directed Sequencing Library Type, select

the X icon under the Actions column.

11. To modify the listed amplicon(s) for Non-Directed Sequencing Library Type, select

the pencil icon under the Actions column.

An Edit Amplicon dialogue box appears. Modify the displayed fields as needed.

Select Confirm.



Figure 5-6 Edit Amplicon for Non-Directed Sequencing Library Type

12. To add an amplicon to the Non-Directed Sequencing Library Type, select Add

Amplicon.

An Add Amplicon dialogue box appears. Define listed fields as necessary. Select

Confirm.

NOTE


Figure 5-7 Add Amplicon for Non-Directed Sequencing Library Type

13. To modify Library Type, select Primer-Directed Sequencing from the dropdown.

A Delete Targets dialogue box appears. Select Confirm.



Figure 5-8 Selecting Primer-Directed Sequencing

14. To define amplicons for Primer-Directed Sequencing, select Add Amplicon.

An Add Amplicon dialogue box appears. Define listed fields as necessary. Select

Confirm.

NOTE


Figure 5-9 Add Amplicon for Primer-Directed Sequencing Library Type

15. Continue to select Add Amplicon until all amplicons have been added.

16. To remove the listed amplicon(s) for Primer-Directed Sequencing Library Type, select

the X icon under the Actions column.



17. To modify the listed amplicon(s) for Primer-Directed Sequencing Library Type, select

the pencil icon under the Actions column.

An Edit Amplicon dialogue box appears. Modify the displayed fields as needed.

Select Confirm.

18. After all desired changes have been made, select Save.

NOTE

The software trims primer sequences for the ForenSeq mtDNA (tiled primer

design). This allows unambiguous interrogation of mtDNA amplicon inserts

(without ForenSeq degenerate oligonucleotides). The mtDNA Variant Processor

v1.0.0 application in BaseSpace does not trim primers from amplicons generated

from a tiled-primer design.

Changing the Default Analysis Method

1. Select Settings.


3. Select an Analysis Method from the dropdown.

4. Select the star icon next to the name of the selected Analysis Method.

The star icon becomes blue and signifies that the default Analysis Method has

changed.

NOTE

There can only be one default Analysis Method per Assay Type.

Figure 5-10 Select New Default Analysis Method

Deleting an Analysis Method

1. Select Settings.


3. Select an Analysis Method from the dropdown.

4. Select Delete next to the name of the selected Analysis Method.

A confirmation box appears.



5. Select Confirm.

Figure 5-11 Delete Analysis Method



CODIS Report Defaults

The following CODIS information can be defined on the CODIS Report Defaults page:



Submission User ID

CODIS Specimen Categories

Defining CODIS Defaults

1. Select Settings.

2. Select CODIS Report Defaults.

CODIS Default Settings are displayed.

3. Enter defaults for the following information:



Submission User ID

4. Select Save.

Figure 5-12 CODIS Report Defaults

Enabling and Disabling CODIS Specimen Categories

1. Select Settings.


3. Select Specimen Categories.

A list of CODIS Specimen categories is displayed. All categories are defaulted for

selection.



4. To deselect any Specimen Category, modify the corresponding toggle button.

Figure 5-13 CODIS Specimen Categories

Creating Custom CODIS Specimen Categories

1. Select Settings.



4. Enter a New Specimen Category Name.

5. Select Add.

The entry appears in the Custom column.



Figure 5-14 Creating Custom CODIS Specimen Categories

Editing Custom CODIS Specimen Categories

1. Select the pencil icon next to the Specimen Category to be edited.

2. Enter the modified Specimen Category.

3. Select the check mark to save the modified Specimen Category.

4. Select the X to cancel.

Figure 5-15 Editing Custom CODIS Specimen Categories



Deleting Custom CODIS Specimen Categories

1. Select the delete icon next to the Specimen Category to be deleted.

The Specimen Category is automatically deleted.

Enabling and Disabling Custom CODIS Specimen Categories

1. Select Settings.



A list of Custom CODIS Specimen Categories is displayed. All categories are

defaulted for selection.

4. To deselect any Specimen Category, modify the corresponding toggle button.



User Management

Enabling and Disabling Accounts

1. Select Settings.

2. Select Admin Settings.

3. To enable a user, the toggle button next to Enabled becomes blue when selected.

4. To disable a user, the toggle button next to Enabled becomes gray when selected.

NOTE

Created accounts are disabled by default. To enable a new account, a user with

Administrator access must follow the above steps to grant access to each new

account.

Figure 5-16 Enabling and Disabling Accounts

Assigning Account Access Levels

1. Select Settings.

2. Select Admin Settings.

3. To enable Administrator access, the toggle button next to Administrator becomes blue

when selected.

4. To disable Administrator access, the toggle button next to Administrator becomes

gray when selected.



NOTE

Administrator access for created accounts is disabled by default. To grant

Administrator access to a new account, a user with existing Administrator access

must follow the above steps for each created account requiring Administrator

access.

Changing an Account Password

1. Select Account from the sidebar menu.

Figure 5-17 Manage Account

2. Select Manage.

A Manage page appears.



Figure 5-18 Change Password

3. Enter Current Password

4. Enter New Password

5. Confirm Password

6. Select Save.



Troubleshooting

Introduction ......................................................................................................... 105 Troubleshooting ForenSeq Runs ........................................................................ 106 Troubleshooting Analysis Errors ......................................................................... 109



Introduction

This chapter features troubleshooting tips for ForenSeq sequencing Runs and analysis errors.

Most of the troubleshooting recommendations for ForenSeq sequencing Runs include library

prep steps, which are detailed in the ForenSeq mtDNA Control Region Kit Reference Guide (document

# VD2019001).



Troubleshooting ForenSeq Runs

Table 13 ForenSeq Run Issues

Problem Resolution

Sample Intensity

(Number of Reads)

Low

If Sample intensity for a Sample is low, this condition can indicate a problem

with Sample quantity or Sample quality.

• Make sure that DNA concentrations are not below the targeted amount.

• Depending on the source, purify the DNA or dilute, and then sequence the

Sample again.

• Load fewer Samples and sequence the Samples again. Make sure to

maintain a minimum of 8 Samples in the Run.

Cluster Density

High

Review the Run Metrics tab. If cluster density for the Run is high, but reads

are above the Sample read count threshold in the Sample Representation tab,

proceed with analysis.

If there is insufficient data for analysis, it can be due to one of the following

reasons.

• Sample from the Purified Library Plate (PLP) or the Library Normalization

Beads 1 (LNB1) reagent was over-pipetted during normalization.

− If the Sample has a high number of alleles with quality control

indicators, then reRun the Sample on a new sequencing Run, and

confirm that correct volumes and reagents are used for normalization.

− If the volume of Sample used is correct, consider further diluting the

PCR product in the Pooled Normalization Libraries (PNL) tube.

− If normalization volumes or reagents were not correct, redo the

Normalize Libraries process and resequence.

• Too much adapter dimer in library prep.

− Run 1–2 µl of the PCR product in the PLP plate on a Bioanalyzer or

Fragment Analyzer and check the amount of primer dimer. Contact

Technical Support with any questions.

− If there is more than 5 % adapter dimer in the PCR product you

check, redo the Purify Libraries process on the remaining

PCR product in the PLP plate using the 30 µl remaining in each well.

Adjust the Purify Libraries process to use 30 µl SPB. Add 32.5 µl

Resuspension Buffer (RSB) and transfer 30 µl to a new 96-well PCR

plate. Proceed with the Normalize Libraries process.

− [Optional] If SPB are limited, purify the PCR product in the PLP

plate using a standard column cleanup method. Use manufacturer

instructions.

• Not enough Hybridization Buffer (HT1) added, or too much normalized

library added to HT1 when preparing libraries. If the Sample has a high

number of alleles with quality control indicators, then reRun the Sample,

on a new sequencing Run, making sure the normalized library is

sufficiently diluted.



Problem Resolution

Cluster Density

Low

Review the Run Metrics tab. If cluster density for the Run is low, but reads

are above the Sample read count threshold in the Sample Representation tab,

proceed with analysis. If the Sample has an insufficient number of reads due

to low density, it is likely due to one of the following reasons:

• During library prep, DNA input was too low or overly degraded, or the

DNA dilution was incorrect. If possible, repeat library prep with more

DNA.

• During library prep, a critical reagent was not added. Repeat library prep.

• LNB1 was not pipetted sufficiently during normalization. Consider

diluting the PCR product in the PNL tube less.

• HT1 was over-pipetted when preparing libraries. Repeat sequencing using

the correct volume.

• Excessive Sample was lost during SPB purification. Repeat library prep

using bead-handling best practices.

• Sample not denatured properly.

− Make sure that the final library pool is denatured with HP3.

− Make sure that the HP3 pH is above 12.5.

− Make sure that the final library pool is heat denatured.

• Instrument blockage.

− Perform a maintenance wash and repeat sequencing.

− Make sure to refill the wash tray and bottle for every wash.

− Make all MiSeq FGx wash solutions daily.

Percentage of

Clusters Passing

Filter Low

If the percentage of clusters passing filter is too low, it is likely due to one of

the following reasons:

• Cluster density is too high. See troubleshooting for Cluster Density High.

• Phasing or prephasing is too high.




• If the problem is still not resolved, it could be that reagents are not

performing as expected. Contact Technical Support.

Phasing or

Prephasing High

If phasing or prephasing is too high, it is likely due to one of the following

reasons:


• Instrument blockage.






Run Does Not

Complete

If the sequencing Run stops before completion, it is likely due to one of the

following reasons:

• When preparing libraries, HSC was not added. Add HSC according to the

Denature and Dilute Libraries process and repeat the sequencing Run.


• If an error message is displayed, contact Technical Support.



Problem Resolution

Read 1 or Read 2

Quality Scores Low

If quality scores for Read 1 or Read 2 are too low, it is likely due to one of the

following reasons:

• Phasing and prephasing are too high. See troubleshooting for Phasing and

Prephasing High.




Index 1 or Index 2

Quality Scores Low

If quality scores for Index 1 or Index 2 are too low, it is likely due to one of

the following reasons:

• Phasing and prephasing are too high. See troubleshooting for Phasing and

Prephasing High.


• There was low diversity in the Index Read because not enough Samples

were sequenced. Repeat sequencing with a minimum of eight Samples.



Reads Per Sample

(Intensity) Low

Review the Sample Representation tab. If the Samples are above the Sample

read count threshold, then proceed with analysis. If reads per Sample are

below the Sample read count threshold, it is likely due to one of the

following reasons:

• During library prep, DNA input was too low, or the DNA dilution was

incorrect. If possible, repeat library prep with more DNA.

• During library prep, a critical reagent was not added. Repeat library prep.

• LNB1 was not pipetted sufficiently during normalization.

− If the Sample has a high number of alleles with quality control

indicators, then reRun the Sample on a new sequencing Run.

− If the Sample has a low number of alleles with quality control

indicators, consider diluting the PNL less and reRun the Samples on a

new sequencing Run.

− If the Run does not pass, redo the Normalize Libraries process.

• HT1 was over-pipetted when preparing libraries. Redo the Normalize

Libraries process.

• The PCR product was not heat-denatured when preparing libraries.

Perform the denature heating step on the Diluted Normalized Libraries

(DNL) tube and repeat the sequencing Run.

Reads Per Sample

(Intensity) for

Human Sequencing

Control Low

Review the Sample Representation tab. If the Samples are above the Sample

read count threshold, then proceed with analysis. If reads per Sample are

below the Sample read count threshold, it is likely due to one of the

following reasons:

• When preparing libraries, HSC was not added. Add HSC according to the

Denature and Dilute Libraries process and repeat the sequencing Run.

• HT1 was over-pipetted when preparing libraries. Repeat the sequencing

using the correct volume.

• The Sample was not heat-denatured when preparing the Sample for

sequencing. Perform the denature heating step and repeat the sequencing

Run.

• HSC was not denatured with HP3 when preparing the Sample for

sequencing. Make sure that the HSC is denatured with HP3 before adding

it to the library and repeat the sequencing Run.



Troubleshooting Analysis Errors

Table 14 Analysis Errors

Problem Resolution

Analysis Error

Message

When you click Create New Analysis, and you receive an analysis error

message, it is likely due to one of the following reasons:

• The ForenSeq Analysis service is not Running.

− On the ForenSeq Universal Analysis Software server desktop, click the

Windows icon in the lower-left corner of the screen.

− Enter Services to find the Services application.

− Open the Services application.

− Locate ForenSeq Analysis in the list of services.

− If the status of the service is not Running, right-click on the service and

select Start.

− After the service has started, try to the Create New Analysis command

again.

• The ForenSeq Analysis service is Running, but the system cannot locate the

Run data to perform an analysis.

− On the ForenSeq Universal Analysis Software server, open the log file

for the analysis service at

(C:\Illumina\Forenseq UAS\Analysis\logs\application.log).

− Find the message, EXCEPTION OCCURRED:System.Exception in the

log file to confirm that the system cannot locate the Run data.

− If the Run repository is stored on a networked location, confirm that

the network is Running.

− Navigate to the Run folder listed in the log file as the RunstoragePath

to confirm that the data exists in the expected location.

− Try the Create New Analysis command again.

All Q Icons on

Dashboard are

Gray

When all Q icons on the dashboard are gray, and no information is available

when you hover over the icons, this condition can indicate the following:

• The system cannot locate the Run data.

− Click the Windows icon in the lower-left corner of the screen.

− Enter Services to find the Services application.

− Open the Services application.

− Locate ForenSeq Analysis in the list of services.

− If the status of the service is not Running, right-click on the service and

select Start.

• The Run repository is stored on a networked location, and the network is

not connected.

− Navigate to the Run folder described in the configuration file.

− Confirm that data exists in the expected location.

− Confirm that no permission changes were made to access the Run

folder location.



Appendix A Supporting Information

Base Call Codes ................................................................................................. 111 Human Sequencing Control Loci ........................................................................ 113 Nuclear mtDNA Insertions (NUMTs) ................................................................... 114 Coverage of the HVII C-stretch ........................................................................... 115



Base Call Codes

The ForenSeq software supports variant calling for more than one nucleotide at an mtDNA

position. When more than one nucleotide exceeds the interpretation threshold, the software

reports the IUPAC code as the consensus for the position.

Table 15 Variant Base Call Codes5

Code Base

A Adenine

C Cytosine

G Guanine

T Thymine

R A or G

Y C or T

S G or C

W A or T

K G or T

M A or C

B C or G or T

D A or G or T

H A or C or T

V A or C or G

When data for the indel and a base call at a position exceed the interpretation threshold, the

app reports the ambiguous call code for an indel as the consensus for the position.

Table 16 Ambiguous Variant Base Call Codes

Code Base

a Adenine

c Cytosine

g Guanine

t Thymine

r a or g

y c or t

s g or c

w a or t

5 Codes are from the International Union of Pure and Applied Chemistry (IUPAC)



k g or t

m a or c

b c or g or t

d a or g or t

h a or c or t

v a or c or g

n Any base

For example, the figure below shows the presence of an ambiguous deletion at position 501.

Because there are an equal number of reads that support a deletion call and a reference call of

G, the app reports an ambiguous call of g as the consensus.

Figure 6-1 Ambiguous Variant Base Calls



Human Sequencing Control Loci

The following is a list of loci that are contained in the sequencing control.

Table 17 Human Sequencing Control Loci

HSC Loci

D3S1358

D5S818

D7S820

D7S820

D8S1179

D13S317

D16S539

D16S539

D18S51

FGA

PentaD

PentaE

TPOX

DYS391

DYS392

DYS460

DXS7423

DXS10103



Nuclear mtDNA Insertions (NUMTs)

Nuclear DNA sequences that are homologous to mitochondrial DNA may be referred to as

Nuclear MTDNA insertions or NUMTs6. During library preparation, these sections may be

enriched alongside their respective mitochondrial DNA targets. While performing alignment,

the ForenSeq UAS identifies reads that map closely to sequences contained in the Reference

Human NumtS compilation7 and removes these reads prior to downstream analysis.

6 D. Mishmar, et al., Hum. Mutat. 23 (2004) 125 – 133. 7 Calabrese, F. M., Simone, D., & Attimonelli, M. (2012). Primates and mouse NumtS in the

UCSC Genome Browser. BMC bioinformatics, 13 Suppl 4(Suppl 4), S15. doi:10.1186/1471-2105-

13-S4-S15



Coverage of the HVII C-stretch

Coverage of the C-stretch (position 303 – 315) in hypervariable region II (HVII) is achieved in

the ForenSeq mtDNA Control Region kit with primer pairs that amplify the region between

positions 262 and 353. Sequences generated from the forward template strand have high

accuracy and alignment. Sequences generated from the reverse template strand have high

accuracy and alignment until the C-stretch is reached.89 Therefore, reads from the reverse

strand, that begin sequencing at position 262, and don’t meet alignment requirements are soft-

clipped in the ForenSeq UAS after position 303; those positions are not used for base calling.

As a result, approximately half of the coverage (reads counts) are obtained for positions 304 to

353, relative to positions 262 to 303 (on the negative strand).

Figure 6-2 HVII C-stretch: Sample Details of ForenSeq mtDNA Positive Amplification Control (HL-

60, 100 pg gDNA) show similar Strand Depth and Total Read Counts at positions 262-303, and at

304-353. INSET: Similar coverage was observed across positions 262-303, where reads from both

strands are reported. Total reads for positions 304 to 353 may be observed at approximately half

that of positions 262-353.

8 Nakamura K, Oshima T, Morimoto T, Ikeda S, Yoshikawa H, Shiwa Y, Ishikawa S, Linak M,

Hirai A, Takahashi H, Altaf-Ul-Amin M, Ogasawara N, Kanaya S (2011) Sequence-specific

error profile of Illumina sequencers, Nucleic Acids Research, 39(13):e90 9 Allhoff M, Schonhuth A, Martin M, Costa I, Rahmann S, Marschall T (2013) Discovering

motifs that induce sequencing errors, BMC Bioinformatics 14(Suppl 5):S1

forenseq universal analysis software v2.0 reference guide · index cv displayed as a percentage,...

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