innovative ngs library construction technology

Sample to Insight

Innovative NGS library prep methods 1

Innovative QIAseq FX technology for construction of NGS libraries for use with Illumina instruments

Ioanna Andreou Ph. D.

Senior Scientist, NGS Life Sciences Team, QIAGEN

Sample to Insight

2

Overview of NGS technologies and innovative NGS library prep methods

Part 1: Introduction to next-generation sequencing (NGS) technology

Part 2: Innovative NGS library construction technology

Part 3: Advanced NGS library prep for challenging samples

Welcome to a 3-part series: NGS technology and applications

Intro to NGS, 11.30.2016

Sample to Insight


Legal disclaimer

• QIAGEN products shown here are intended for molecular biology

applications. These products are not intended for the diagnosis,

prevention or treatment of a disease.

• For up-to-date licensing information and product-specific

disclaimers, see the respective QIAGEN kit handbook or user

manual. QIAGEN kit handbooks and user manuals are available

at www.qiagen.com or can be requested from QIAGEN

Technical Services or your local distributor.

Sample to Insight


Agenda

1 Introduction

FX technology for NGS library construction

Single cell analysis

Single cell DNA sequencing

Single cell RNA sequencing

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Sample to Insight


Agenda

1 Introduction





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5

Sample to Insight

Innovative NGS library prep methods

NGS technology overview and applications series: Part 2

6

Overview of NGS technologies and innovative NGS library prep methods

Part 1: Introduction to next generation sequencing (NGS) technology



Sample to Insight


Typical challenges in whole genome sequencing

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Faster turnaround

Higher library complexity

Control per-sample costs

Nucleic acid isolation

Data analysis and

interpretation

Library preparation

and QCSequencing

Sample collection

and stabilization

→ Permits process scale-up

→ Maximizes clinical utility of data

→ To preserve sample information content

→ To enable high-quality downstream analysis

→ Time, consumables, first-pass success rate

Sample to Insight


Current options for DNA fragmentation are suboptimal

Mechanical shearing Enzymatic shearing

FragmentDNA

Addadapters

Amplify and QC

library

Typical NGS Library Prep Process

High costs (instrumentation lab space, hands-on-time)

Harder to scale-up Intermediate cleanup steps

Poor data quality, introduction of sequence bias

Inflexible protocol Not easy to adapt for different

DNA input

Sample to Insight


Agenda

1 Introduction





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Sample to Insight


QIAseq FX: Gold-standard quality from an enzymatic workflow

Single-use barcoded adapters can be used all at once or in batches of 10–12

Faster and easier to automate without the need for fragmentation instrumentation

Outperforms other enzymatic shearing procedures on genomic coverage

Flexible fragment size, batch size and input DNA amount

Complete kit that includes reagents for fragmentation, ligation, library amplification and 96-plex dual-barcoded adapters

Suitable for whole genome sequencing, whole exome / hybrid capture, metagenomics

Sample to Insight


QIAseq FX – the ideal balance of speed and high data quality

QIAseq FX uses only a standard thermocyler and 96-well PCR plates for truly scalable library prep 2.5 hour total workflow <20 min hands-on time Single-tube enzymatic reaction workflow Easy manual implementation or adaptation for automated liquid handling QIAGEN high-fidelity library amplification and plate format 96-plex adapters included

Adapter ligation Cleanup

Library amplification

(optional)

Fragmenta-tion, end-

repair and A-tailing

2.5 h

Single tube

Purified gDNA

1 ng – 1 µg

Sample to Insight


PCR-free option from as little as 10 ng input DNA

QIAseq FX (50 ng PCR-Free)QIAseq FX (100 ng PCR-Free)Mechanical shearing (100 ng) with PCRQIAseq FX (100 ng) with PCR

0 20 40 60 80 1000

0.5

1

1.5

2

Excellent G/C coverage with or without PCR

GC% over 100 bp regions

Nor

mal

ized

cov

erag

e

PCR-free library yield (nM) vs. input (ng)

Yields as low as 2 nM can typically be sequenced

10 nM PCR-free yield from just 100 ng input

Sample to Insight

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Compatible with downstream hybrid capture technologies

QIAseq FX

Library preparation:

- gDNA fragmentation

- Barcoded adapter ligation

- Library amplification

Purified gDNA1 ng–1 µg

Library QC


Hybrid capture

Target enrichment

(IDT xGen, Agilent SureSelect)

QIAseq FX library

Sequence selected library

http://eu.idtdna.com/pages/products/nextgen/target-capture#

http://eu.idtdna.com/pages/products/nextgen/target-capture

Sample to Insight

Mechanical-quality fragmentation from an enzymatic workflow


Sample-to-sample fragmentation reproducibility

5 min 10 min0

200

400

600 Frag. #1Frag. #2Frag. #3Frag. #4

Fragmentation time

Ave

rage

frag

men

t siz

e (b

p)

Customize fragment size by adjusting incubation time

5 min 10 min0

200

400

600Input DNA species

Bacterial MixHuman

Fragmentation timeA

vera

ge fr

agm

ent s

ize

(bp)250 bp

450 bp

1000 bp

Sample to Insight

QIAseq FX exhibits less G/C bias than comparable methods


QIAGEN QIAseq FXSupplier N – Enzymatic shearingMechanical shearing + Standard LP(Tagmentation not possible at 100 ng input)

QIAGEN QIAseq FXSupplier N - EnzymaticMechanical shearing + Standard LPSupplier I - Tagmentation

Sample to Insight


0 50 100 1500

10

20

30

40

50

Supplier N - 100 ngA T C G

read position (nt)

% in

divi

dual

nuc

leot

ide

0 50 100 1500

10

20

30

40

50

Supplier I - 1 ngA T C G

read position (nt)

% in

divi

dual

nuc

leot

ide

0 50 100 1500

10

20

30

40

50

QIAseq FX 100 ngA T C G

read position (nt)

% in

divi

dual

nuc

leot

ide

0 50 100 1500

10

20

30

40

50

QIAseq FX 1 ngA T C G

read position (nt)

% in

divi

dual

nuc

leot

ide

QIAseq FX generates a purer random base composition

Sample to Insight

Superior genomic coverage and duplication rate


0 10 20 30 40 50 60 70 80 90 1000

0.02

0.04

0.06

0.08

0.1

0.12

Coverage distribution

Coverage depth (X)

Frac

tion

of ta

rget

gen

ome

QIAseq FX (100 ng)Supplier N – Enzymatic (100 ng)Mechanical shearing + Standard LP (100 ng)Supplier I – Tagmentation (1 ng)

QIAseq FX (1 ng and 100 ng*)Supplier N – Enzymatic (1ng)Mechanical shearing + Standard LP (1 ng)Supplier I – Tagmentation (1 ng)

100n

g

Duplication rate, 1 ng input

Gold-standard

% duplication

Mechan

ical s

heari

ng

Sample to Insight


Agenda

1 Introduction





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Cells differ on the genome level

Genome variations occur in health and disease

(1) Iourov, I.Y. et al. (2010) Somatic Genome Variations in Health and Disease, Curr Genomics 11(6)

Somatic genome variations consist of :

Aneuploidy

Structural rearrangements

Copy number variations

Gene mutations

Somatic genome variations

Occur during normal development/aging

Contribute to pathogenesis

Are the cause of diseases such as cancer, autoimmune, brain and other diseases

Examples (1)

Aneuploidy in pre-implantation embryos occurs in 15–91% of samples

Aneuploidy in skin fibroblasts occurs in adults Middle age: in 2.2% of cells Aged: in 4.4% of cells

Almost all cancers are caused by different types of genome variations including aneuploidy/polyploidy, structural rearrangements, gene amplifications, gene mutations


Sample to Insight


Seemingly identical cells – unique transcriptional patterns

Cells change their transcription pattern:

(1) Kumar L.M. et al. (2014) Deconstructing transcriptional heterogeneity in pluripotent stem cells. Nature 4;516

The transcriptome of a cell is not fixed but dynamic

The transcriptome reflects the Function of the cell Type of the cell Cell stage

Gene expression is influenced by intrinsic or extrinsic factors (signaling response, stress response)

Only on single cell level you obtain: Real (not average) transcriptome gene

expression data Allelic expression data A deeper understanding of the

transcription dynamics within a cell

Heat map of single cell RNA-seq data for selected pluripotency regulators (1)

Sample to Insight

Single cell analysis enables new insights

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CTC = Circulating tumor cells, PGD = Pre-implantation genetic diagnosis

Cellular heterogeneity

Detection and analysis of rare cells (example: CTC from liquid biopsy)

Identification of cell subpopulations based on genomic structure or gene expression (tumors, tissues, immune cells, cell cultures)

Limited availability of cells Analysis of limited sample

material (example: embryo biopsy for PGD, fine-needle aspirates)

ApplicationReason

Biological insights instead of data from an aggregate

No data

Bulk result Single cell data


Devika Mathur - QIAGEN

Images from extranl sources must be referenced

Sample to Insight


Agenda

1 Introduction





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3

4

5

Sample to Insight

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Discover the QIAseq FX Single Cell DNA Library Kit

Maximize coverage

Superior and more uniform genome coverage

compared to other kitsHigh sequence

fidelityMDA-based amplification

technology, proven for higher fidelity compared to

PCR-based methods

Enables biobanking

Excess amplified DNA can be stored for follow-up use, perfect for confirming novel

mutations or structural variants.

Higher diversity libraries

PCR-free workflow, better library diversity by

eliminating PCR-duplicates

Less GC-biasSuperior presentation of

GC-rich regions, perfect for bacteria with high GC-

content genomes

Robust and streamlined

workflowEverything in one package,

single-use adapters cut down on contamination possibilities, no need for

extensive QC

Minimize hands-on-timeUnder 4 h workflow from

single cell to library, without any additional kits

Superior sensitivity

The only kit sensitive enough for single bacterial genomes

QIAseq FX Single Cell

DNA Library Kit


Sample to Insight


For single-cell DNA sequencing

Ideally suited for

The analysis of inter-cellular genome heterogeneity The analysis of aneuploidy and

sub-chromosomal copy number variations Sequence variation analysis (SNV, structural variants) in single cells Whole genome sequencing from rare samples Resequencing or de-novo sequencing of unculturable microorganisms For new type of experiments such as low-pass sequencing, consensus-based

variant calling


DNA Library Kit

Sample to Insight

QIAseq FX Single Cell DNA Library Kit

Complete cell-to-library solution

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Primary sample isolation

Single cell isolation

NGS library construction NGS run Data

analysis InterpretationSample Insight

Single eukaryotic cell Single bacterial cell Picogram levels of purified DNA

Whole genome NGS Library Illumina-compatible Sequence variants Structural variants Aneuploidy Bacterial genomes


Sample to Insight


QIAseq FX Single Cell DNA Library Kit: kit contents

Both kits contain: Cell lysis reagents Enzymes and buffers for whole genome amplification Enzymatic DNA fragmentation Single-step NGS library preparation Single-use, disposable Illumina Adapters in 96-well

format Multiple reagent aliquots to reduce contamination risk

and freeze-thaw cycles

What is not included: AMPure XP beads for library purification PCR reagents for library amplification: not needed as the

entire workflow is PCR-free qPCR reagents for library quantification: recommended

for accurate flow-cell loading

Cat No./ID: 180713QIAseq FX Single Cell DNA Library Kit (24)

Cat No./ID: 180715QIAseq FX Single Cell DNA Library Kit (96)

Sample to Insight


QIAseq FX Single Cell DNA Library Kit: the workflow

Cell lysis From single eukaryotic or bacterial cells, or small amounts (pg–ng) of intact gDNA Starting with 4 µl cell material in PBS (included) Prepare lysis buffer, mix with cells, incubate for 10 minutes at 65°C. If using purified DNA as

input, incubate for 3 minutes at room temperature Hold at 4°C

Sample to Insight



Whole genome amplification Prepare WGA master mix, mix with lysed cells, incubate for 2 h

Amplified gDNA can be used directly or frozen until needed There will be an excess of amplified gDNA, this can be stored for later use or follow-up

studies (i.e. confirming deletions detected with NGS via PCR or Sanger sequencing) Library preparation accepts a wide range of inputs, so quantification of the amplified DNA

is not needed

Sample to Insight



NGS library preparation Prepare FX master mix, add to diluted WGA product and incubate for ~15 min. Insert size

can be set by user. Hold at 4°C Add adapters from single-use adapter plate Prepare ligation master mix, add to samples and incubate for 15 min to produce library

Cell lysis15 min

WGA2 h

FX library preparation

70 min

Purification20 min

ILLUMINA sequencing

3h 45 min with ~40 min hands-on time

Sample to Insight



Library purification Remove excess adapters with double-sided Agencourt AMPure XP cutoff

No PCR amplification necessary: protocol generates sufficient library without enrichment Library quantification via qPCR (i.e. QIAseq Library Quant) is highly recommended to

ensure accurate clustering on sequencer

Cell lysis15 min

WGA2 h


70 min

Purification20 min

ILLUMINA sequencing

3h 45 min with ~40 min hands-on time

Sample to Insight

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Discover complete genome coverage

Perfect for low-pass sequencing

Don’t miss out on variants or structural features due to low coverage or locus

drop-outs

See more of the genome with the same sequencing

depth


Libraries generated from single PBMC using the QIAseq FX DNA Library Kit or kits from two other suppliers and sequenced at low depth using MiSeq. Data were analyzed according to Zhang C.Z. et. al “Calibrating genomic and allelic coverage bias in single cell sequencing“, (2015) Nat. Commun. 6, 6822.

Comprehensive genome coverage

Genome coverage of various kits

Sample to Insight

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More uniform coverage, even with GC-rich regions

Perfect for bacteria with high GC-

content genomes

Coverage of traditionally

difficult-to-sequence regions


Coverage versus GC content

Libraries were generated from single PBMC using the QIAseq FX DNA Library Kit or kits from two other suppliers and sequenced at low depth on the MiSeq. Data was analyzed using the CLC Genomic workbench 8.5.1.

Sequence GC-rich regions

Sample to Insight

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Highest fidelity sequence data: Have confidence in your results

Perfect for low-pass consensus variant

calling

Lower background when analyzing

sequence variants or mutations and

small indels

Fewer spurious sequence errors in

your dataset

Fewer false positives


Single cell libraries from isolated PBMCs were sequenced with an Illumina MiSeq. Reads were mapped to the human genome (hg19) and sequence mismatches between NGS data and the reference were computed using the CLC Genomic workbench 8.5.1. Data plotted are the mean proportion of sequence differences +/– standard deviation for 3 replicates.

Combined error-rate of several single cell NGS methods Highest sequence

fidelity

Sample to Insight

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Analyze copy number variants and aneuploidy

Biobanking allows follow-up

experiments: confirm structural

variations with PCR or Sanger

sequencing

With comprehensive coverage, detect structural variants

regardless of where they are in the

genome


QIAseq FX Single Cell DNA libraries from PBMCs and Jurkat cells were sequenced to 0.1x depth on a MiSeq. Reads were mapped to human genome (GRCh38) and the copy number variation of Jurkat vs PBMCs (control diploid cells) was assessed using the methods published in: Chao Xie, Martti T Tammi, “CNV-seq, a new method to detect copy number variation using high-throughput sequencing”, BMC Bioinformatics, 2009,10:80. The plot is the Log2 ratio (Jurkat/PBMC) of coverage using a window size of 500 Kb for chromosome 2 from a cell with an approx. 25 Mbp deletion.

Detection of a 25 Mbp deletion in a single cellAnalyze

CNVs and aneuploidy

Sample to Insight

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High-yield WGA enables biobanking and confirmatory testing

Follow-up tests to confirm structural

variants or sequence variants

or novel discoveries

Store unused amplified gDNA at

–20 for later use


Data from 4 individual PBMCs for each kit, with the kit from Supplier R, no extra DNA was available for storage.

Yields of amplified gDNA of various kits Biobanking enables follow-up

testing

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Robust FX accepts a wide range of inputs: Save time on QC

PCR-free libraries from a wide range of

amplified gDNA inputs

Go directly from amplified gDNA to library prep: simple

dilution for all samples

No need to quantitate amplified gDNA: save time on

QC


In this experiment, libraries were prepared from different amounts of the same pool of amplified gDNA.The library yield after the final purification is shown. Any libraries over 2 nM concentration can be sequenced directly and do not need PCR amplification.

Consistent results with

less QC

Yields of generated library vs DNA input

Sample to Insight

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Highly tunable fragmentation: Determine insert size

Adjust insert size with longer or

shorter FX incubation

Insert size consistent with

varying amounts of amplified gDNA


Libraries with a varying amount of amplified gDNA were prepared and subjected to a gradient of FX fragmentation times. The plots show that the longer the incubation times, the shorter the inserts, and that this approximately follows the same trend regardless of the amount of input used.

Mean insert size per incubation timeInsert size

determined by fragmentation

incubation time

Sample to Insight


Agenda

1 Introduction





2

3

4

5

Sample to Insight

Discover the QIAseq FX Single Cell DNA Library Kit

39

Maximize transcript discovery

Discover a greater number of transcripts with the same

sequencing depth

High sequence fidelity

High-fidelity WTA minimizes spurious sequence errors.

Ideal for viral RNA sequencing.

Enables biobanking

Excess amplified DNA can be stored for follow-up use,

perfect for confirming novel mutations or structural

variants

Higher diversity libraries

WTA technology with less dropouts and less length-

bias against long transcripts and highly efficient library

preparation with maximized conversion rate

No PCR duplicatesPCR-free workflow

eliminating PCR duplicates

Robust and streamlined

workflowEverything needed in one

package. Single-use adaptors cut down on contamination

possibilities, no need for extensive QC.

Minimize hands-on-time5.5 h workflow from single cell to library, without any

additional kits

Uncover mRNA and

lincRNASequence

lincRNA and mRNA with a

single protocol


RNA Library Kit


Sample to Insight


For single cell RNA sequencing

Ideally suited for

Sensitive transcript discovery

and differential gene expression analysis from

single eukaryotic cells

Transcriptome analysis with best-in-class transcript detection

The analysis of both mRNA and long non-coding RNAs in a single dataset

Studies in inter-cellular heterogeneity

RNA-seq from limited amounts of difficult-to-obtain samples

Studies in infectious disease research

QIAseq FX Single Cell RNA Library

Kit

Sample to Insight

QIAseq FX Single Cell RNA Library Kit

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Complete cell-to-library solution

Primary sample isolation

Single cell isolation

NGS Library construction NGS run Data

analysis InterpretationSample Insight

Single eukaryotic cell Picogram levels of purified

RNA from different species

Whole genome NGS Library Illumina-compatible Transcript discovery Gene expression Differential expression Viral RNA


Sample to Insight


QIAseq FX Single Cell RNA Library Kit: the contents

Both kits contain: Cell lysis and gDNA degradation reagents Reverse transcription primers, buffers and enzyme Enzymes and buffers for cDNA amplification Enzymatic cDNA fragmentation Single-step NGS library preparation Single-use, disposable Illumina adapters in 96-well

format Multiple reagent aliquots to reduce contamination risk

and freeze-thaw cycles

What is not included: Agencourt AMPure XP beads for library purification PCR reagents for library amplification: not needed as the

entire workflow is PCR-free qPCR reagents for library quantification: recommended

for accurate flow-cell loading

Cat No./ID: 180733QIAseq FX Single Cell RNA Library Kit (24)

Cat No./ID: 180735QIAseq FX Single Cell RNA Library Kit (96)

Sample to Insight


QIAseq FX Single Cell RNA Library Kit: the workflow

Cell lysis From single eukaryotic or small amounts (pg – ng) of high-quality, purified RNA. Starting with 7 µl cell material in PBS (included). Prepare lysis buffer, mix with cells, incubate for 8 minutes. Cool to 4°C. Add gDNA degradation reagent, incubate for 10 min.

Cell lysis15 min

WTA3 h 45 min


70 min

Purification20 min

Illumina sequencing

5.5h with ~1h hands-on-time

Sample to Insight



Whole transcriptome amplification Prepare RT master mix, mix with lysed cells, incubate 1 h Prepare cDNA ligation mix, incubate for 30 min Prepare cDNA amplification mix, mix with unamplified cDNA, incubate for 2 h

Amplified cDNA can be used directly or frozen until needed. There will be an excess of amplified cDNA, this can be stored for later use or follow-up studies (i.e.

confirming deletions detected with NGS via PCR or Sanger sequencing). Library preparation accepts a wide range of inputs, so quantification of the amplified cDNA is

generally not necessary.

Cell lysis15 min

WTA3 h 45 min


70 min

Purification20 min

Illumina sequencing


Sample to Insight



Cell lysis15 min

WTA3 h 45 min


70 min

Purification20 min

Illumina sequencing


NGS library preparation Prepare FX master mix, add to diluted WTA product and incubate for ~15 min. Insert size can

be set by user. Hold at 4°C. Add adapters from single-use adapter plate. Prepare ligation master mix, add to samples and incubate for 15 min to produce library.

Sample to Insight



Library purification Remove excess adapters with double-sided Agencourt AMPure XP cut-off

No PCR amplification necessary: protocol generates sufficient library without enrichment. Library quantification via qPCR (i.e. QIAseq Library Quant) is highly recommended to

ensure accurate clustering on sequencer

Cell lysis15 min

WTA3 h 45 min


70 min

Purification20 min

Illumina sequencing


Sample to Insight

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Discover more: higher library diversity than competing workflows

See more of the transcriptome

Discover a greater number of

transcripts with the same sequencing

depth

Spend reads sequencing RNA

molecules, not PCR duplicates


Libraries were generated from 100 pg of input from the same pool of RNA isolated from PBMCs with either the QIAseq FX Single Cell RNA Library Kit or a commonly used workflow employing separate kits for whole transcriptome amplification and library preparation from Suppliers C/I. Libraries were multiplexed and sequenced on the same MiSeq to approximately the same sequencing depth. After QC and mapping, the number of annotated transcripts with TPM >1 was computed. Data were then rarified repeatedly, where a subset of reads was selected at random, and the number of annotated transcripts with TPM >1 was computed for each sub-sampled set of reads. This was repeated at multiple read depths.

Discovery plot High library diversity

Sample to Insight

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Discover more: high library diversity from single cells

Usually, single cell libraries are easily

saturated

Sequence more, discover more


Libraries were generated from single isolated PBMCs the QIAseq FX Single Cell RNA Library Kit. After QC and mapping, the number of annotated transcripts with TPM >1 was computed. Data were then rarified repeatedly, where a subset of reads was selected at random, and the number of annotated transcripts with TPM >1 was computed for each sub-sampled set of reads. This was repeated at multiple read depths.

High library diversity: even

from single cells

Discovery plot

Sample to Insight

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Discover more: high number of transcripts detected

High diversity libraries maximize transcript detection in each single cell


Libraries were generated from 3 single isolated HeLa cells or from 100 pg of bulk RNA isolated from the same cells using the QIAseq FX Single Cell RNA Library Kit. After QC and mapping, the number of annotated transcripts with FPKM>1 was computed.

Maximize transcript detection

Number of transcripts

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High dataset diversity provided by PCR-free library preparation

PCR-free workflow eliminates

PCR duplicates

Frees read-depth for discovering new

transcripts


Libraries were generated from single isolated PBMCs using the QIAseq FX Single Cell RNA Library Kit and a kit from Supplier C. Libraries were multiplexed and sequenced on the same run of a MiSeq instrument to equal sequencing depth. Duplicates were calculated using FastQC. PCR duplicates represent reads that provide no additional insight into the sample, and detract from the sensitivity of the experiment by consuming valuable sequencing depth.

Duplicate levelSequence

new transcripts not PCR

duplicates

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Uncover linc and mRNA with a single technique

Sequence mRNA and lincRNA in a single experiment

Examine protein-coding gene

expression and regulatory RNAs simultaneously


Libraries were prepared from single PBMCs or HeLa cells using the QIAseq FX Single Cell RNA Library Kit or a commonly used workflow employing separate kits for whole transcriptome amplification and library preparation from Suppliers C/I. Libraries were multiplexed and sequenced on the same MiSeq to approximately the same sequencing depth. After QC and mapping, the proportion of reads mapping to annotated lincRNA for each library was calculated.

Sequence lincRNA and mRNA with a

single protocol

% of detected lincRNA

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High proportion of mRNA reads

Discover the insights only a

combined mRNA and lncRNA

dataset can bring

Use single cell sensitivity to

analyze mRNA expression


Libraries were prepared from single isolated PBMCs. The QIAseq FX Single Cell RNA Libraries were multiplexed and sequenced on an Illumina NextSeq. After QC and mapping via STAR, the proportion of reads mapping to each GO annotation was calculated with HTSeq.

Maintains a high proportion

of protein-coding reads

RNA biotypes

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Consistent, robust cDNA amplification enables biobanking

Protocol consistently

generates excess cDNA

Consistent WTA produces consistent

libraries

Freeze excess amplified cDNA for follow-up studies or confirmatory testing


Whole transcriptome amplification yield from single cells from the QIAseq FX Single Cell RNA Library Kit and a competing, PCR-based method (Supplier C). 8 Replicates from different single cells are shown.

Yield of amplified cDNA in ng Robust WTA enables

biobanking

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Summary

QIASeq FX Kits for NGS library construction use an innovative technology that:

Uses a fast and streamlined workflow, without the need of additional kits and highly

specialized instrumentation

Delivers high yields of high-quality libraries ready for sequencing on Illumina platforms

Addresses different applications

DNA libraries from a wide range of input DNA

PCR-free DNA libraries from single eukaryotic and bacterial cells and very limited purified DNA

material

PCR-free RNA libraries from single eukaryotic cells, limited purified RNA material and viral RNA

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“Overview of NGS technologies and innovative NGS library prep methods”

Part 1: Introduction to next-generation sequencing (NGS) technology




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