ion reproseq pgs kits for the ion genestudio s5 systems · 2020-05-12 · • rapid—sample to...

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Colin Davidson, PhD

Ion ReproSeq PGS Kits for the Ion GeneStudio S5 Systems

For Research Use Only. Not for use in diagnostic procedures.

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Global Impact ─ Countries with Laboratories Using Ion ReproSeq PGS Kits

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Complete Solution for Aneuploidy Detection by Low-Pass Genome Sequencing

Starting from a biopsy sample of 1–10 cells

Library prepCombined DNA extraction,

whole genome amplification (WGA), and sample barcoding

Template prepAutomated clonal

amplification of libraries

SequencingSimple, cartridge-loaded

reagents and user interface

AnalysisAutomated

Ion Reporter™ software

&

• Rapid—sample to answer typically in <10–13 hours

• Flexible—multiplexing of 16–96 samples/run

• Reliable—mosaicism detection and calling

• Simple—plug-and-play instrumentation


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• Like whole genome sequencing (WGS) but on a diet• WGS requires sufficient number reads (~360M reads*) to cover the entire genome at adequate

depth to genotype• However, to detect large chromosomal and sub-chromosomal CNVs, sequencing the genome

requires much lower depth (<0.01X coverage per sample or ~100,000 reads)

What is Low-Pass Genome Sequencing?

*30X coverage with 2X125 bp PE reads

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• Bias has many sources including library prep (WGA), sequencing (GC content), and alignment (repetitive regions)

• For detecting CNVs, a baseline is used to overcome bias• Reads in a sample are compared to normal controls• Corrections are made for reproducibly over- or under-

representation of sequencing reads• Aneuploidies are called from data outside this normal range

• It’s all about the base(-line)• No need to run a control sample each time• A baseline is made from many controls runs helps:

• Normalize for GC and other commonly observed biases• Correct for run-to-run differences

• In Ion Reporter, baselines are provided or can be user-generated

Reproducible Bias in Low Pass Genome Sequencing

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Ion ReproSeq PGS Kits for the Ion GeneStudio S5 Systems

Three kit configurations support sample scalability of 16, 24, and 96 samples per run

Detection of whole-chromosome aneuploidies and chromosome-arm copy number events in as little as 10 hours

Ion SingleSeq™ library kit includes reagents to extract, amplify, and prepare barcoded libraries from a single cell

Ion 520™ Chip24 samples/run



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Ease of Use Through Single-Use Consumables and Easy Tracking with RFID Tags


Large HD touchscreen

Temperature-controlled chip clamp

Wash solution and waste cartridge(behind W2 solution)

Automated cleaning solution

Sequencing reagent cartridge• Automated template prep and chip loading

• 15 minutes total setup time for template prep

Ion GeneStudio™ S5 SystemsIon Chef™ System

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Total Turnaround Time <10–13 Hours from Cells to Data for 16–96 Samples


Ion ReproSeq PGS Kit with Ion 520 Chips (24 samples/run)



3.1

3.5

4.5

5

5

5

1.25

1.5

2.25

0.25

0.5

1

0 2 4 6 8 10 12 14

16 samples/run

24 samples/run

96 samples/run

Time (hr)

Library prep Template prep Sequencing Analysis

Turnaround time: sample to analyzed results with the Ion ReproSeq PGS Kits on the Ion GeneStudio S5 Plus System

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2.5

1.75

2

3

0 2 4 6 8 10 12 14

Illumina VeriSeq™ PGS Kit (24 samples)**

Ion ReproSeq PGS Kit with Ion 510 Chips (16 samples/run)*



Hands-on time Total time

Rapid, Scalable, Cost-Effective, and Simple PGS Workflow on a Single Platform

* Total time on the Ion GeneStudio S5 Plus System** Values obtained from VeriSeq PGS Kit by Illumina presentation slides


Time (hr)

Comparison of hands-on and total turnaround time for aneuploidy analysis using the three Ion ReproSeq PGS Kits for the Ion GeneStudio S5 Plus System and the Illumina VeriSeq PGS kit

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Simplified Bioinformatics


Ion Reporter Software

Tunable aneuploidy detection with preconfigured analysis workflows

Cloud or local server options

For Ion Reporter Software v5.4 or later • New mosaicism analysis• Gender masking• Improved smoothing/plots

For Ion Reporter Software v5.10 or later • Custom (improved) sensitivity with transition penalty • Smaller tile size baselines for small event calling• Customizable filter of mosaic events around normal ploidy Cloud-enabled

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Aneuploidy Analyses Using Different Ion Reporter Software Aneuploidy Workflows

Top three plots are courtesy of Genoma Group Italy. Bottom plot contains data from a Coriell sample.

Smoothing

Default Ion ReproSeqworkflow

No smoothing

Gender masked(reported normal)

Mosaicism Mixture of a normal female, with male (chr9 duplication, 45 Mbp) at 40%

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Internal Testing Results For Non-mosaic Events

>99% average sensitivity and specificity for 5-cell inputs fordeletions and duplications ≥17 Mbp

Sample No. of cells

No. of libraries

Average total

reads/ barcode

Average MAPD

Average sample

sensitivity

Average chromosome

specificity

45 Mbp chr 9 duplication and48 Mbp chr 13 deletion

5 cells 766 127,447 0.186 99.74% 99.96%

21 Mbp chr 16 duplication and17 Mbp chr 2 deletion

5 cells 96 545,875 0.109 100%‡ 100%‡

5 cells 96 100,000* 0.193 98.96%‡ 100%‡

‡Transition Penalty = -2.0; ≥ 0.1 confidence*downsampledMAPD≤0.3

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Ion Reporter Feature Improvements ─ Transition Penalty Parameter Adjustment

• Improve detection of small segmental CNV events• Decrease sensitivity by penalizing ploidy changes (higher negative number)

• High Sensitivity = -3• Medium Sensitivity = -5• Low Sensitivity = -15

Workflow Parameters chevron, CNV Finding, Main tab, CUSTOM sensitivity

Workflow Parameters chevron, CNV Finding, Advanced Tab, CNV Transition Penalty = -2

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Transition Penalty Adjustment Increases Sensitivity Calls 7.9 Mbp Deletion

High Sensitivity (Trans. Pen. = -3)

Custom Sensitivity (Trans. Pen. = -2)

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Ion Reporter Feature Improvements ─ Expected Normal Ploidy Buffer (ENPB)

• Improve detection of mosaic CNV events• Mosaic workflows can have false positive calls near the expected normal ploidy • Expected Normal Ploidy Buffer (ENPB) can filter these putative false positives• Default filter chain: “Aneuploidy Mosaicism”

• ENPB: show < -0.2 or > 0.2 ploidy units• Confidence >= 0.1

• Or set custom ENPB zone and Confidence

Default “Aneuploidy Mosaicism” Create new filter chainEdit thresholds for ENPB, and for confidence threshold

OR

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Increased Sensitivity and Customizable Filtering for Calling Segmental Mosaic Events

Improved calling of a segmental 40% mosaic deletion on chromosome 13

No ENPB Yes ENPB

High sensitivity (TP -3), confidence > 0.1

Custom sensitivity (TP -2.33), confidence > 0.1

Ploidy 1.8

Ploidy 1.65

High sensitivity (TP -3), confidence > 0.1

Custom sensitivity (TP -2.33), confidence > 0.1

New productive reads per sample (mapped, non-duplicate reads - excluding mitochondrial reads and Y chromosome for female samples)

Ploidy 1.65

Customizable noise removal around 2N using the ENBP

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Ion Reporter Feature Improvements ─ Smaller Tile Sizes

• Further sensitivity improvements via custom tile size (default is 2 Mbp)• New baselines provided at 1 Mbp and of 0.5 Mbp (can be user defined)

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Smaller Tile Size Baseline Allows Calling of a 4.5 Mbp Deletion on Chromosome 1

96-plex 150,000 read subsample(custom sensitivity (TP -2); default 2.0 Mbp tile)

96-plex 150,000 read subsample(high sensitivity (default TP-3); 0.5 Mbp tile)

Cell line (GM22991): 46,XX.ish del(1)(p36.32)(CEB108/T7-,SKI-,D1S3739+).arr 1p36.32(742429-5215341)x1

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• Default high sensitivity workflows consistently calls events to 6─8 Mb with detection of smaller events between 2─8 Mb requiring TP sensitivity adjustments and/or smaller baseline tile size adjustments

Default high sensitivity workflows consistently call events to 6─8 Mb

Sample Type No. CNV Event Size Range (Mb) CNV Events Called* (Mb) Cell-line gDNA 7 3.7─18.8 3.7─18.8 MbSorted cell-line cells 15 1.19─100.4 11.9─100.4 MbTrophectoderm embryo biopsy 26 7.8─71.2 7.8─ 71.2 MbEmbryo biopsy WGA product 41 4.4─163 8─163

*transition penalty: -3; baseline tile size 2 Mb

Subchromosomal aberrations from a trophectoderm biopsy sample

25 Mb

8 Mb

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High Resolution Event Calling Using Sensitivity Adjustments

Event Sample Type Transition Penalty Tile Size (Mb) 1.81 Mb Del(9) Sorted cell-line cells -3 0.22.19 Mb Dup(6) Sorted cell-line cells -2 0.52.53 Mb Del(5) Sorted cell-line cells -3 0.53.36 Mb Dup(16) Sorted cell-line cells -3 0.24.4 Mb Dup(22) Embryo biopsy WGA product -3 14.5 Mb Del(1) Cell-line gDNA -3 0.5

Detection of subchromosomal aberrations from a sorted cell line cell; 99 Mb dup(3q12.1q29x3.0) and a 1.81 Del(9p24.3x1.0) CNV events using transition penalty: -3; baseline tile size: 0.2 Mb

99 Mb

1.81 Mb

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Mosaicism Calling and Easier Data Visualization with Smoothing

IR without smoothing

IR with smoothing and calling

Mosaicism in day 5 embryo biopsies - a mixture of normal and aneuploid cells

gDNA mixture - female, normal with male (chr9 duplication, 45Mbp) at 40% background

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Five-cell Mixtures Whole Chromosome Mosaic Profiles Called From 20─80%

20% chr21 2.2N, chrX 1.2N, chY 0.8N

40% chr21 2.4N, chrX 1.4N, chY 0.6N

60% chr21 2.6N, chrX 1.6N, chY 0.4N

100% chr21 3N, chrX 2.0N, chY 0.0N

80% chr21 2.8N, chrX 1.8N, chY 0.2N

Data courtesy of Center for Reproductive Medicine, Weill Cornell Medical College

Cell lines 46, XY and 47, XX, +21 selected under a microscope, and pooled at 0%, 20%, 40%, 60%, 80% and 100%Mosaicism event calling for chr21, chrXand chrY

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• Complete Solution for Aneuploidy Detection by Low-Pass Genome Sequencing• Rapid—sample to answer typically in <10–13 hours• Flexible—multiplexing of 16–96 samples/run• Simple—plug-and-play instrumentation

• Customizable aneuploidy analysis workflows in Ion Reporter software v5.10 or later can be used to improve automated calling of mosaic and subchromosomal CNV events• Mosaic analysis workflows are capable of automatically calling whole chromosome mosaicism ≥20%• Default high sensitivity workflows can consistently call subchromosomal CNV events ≥8 Mb whereas CNV

events from 2─8 Mb can be detected using adjustable custom sensitivity parameters for automated calling

Conclusions

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thermofisher.com/ionreproseq

For Research Use Only. Not for use in diagnostic procedures. © 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Illumina and VeriSeq are trademarks of Illumina, Inc.

ion reproseq pgs kits for the ion genestudio s5 systems · 2020-05-12 · • rapid—sample to...

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