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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
For Research Use Only. Not for use in diagnostic procedures.
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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
Ion 530™ Chip96 samples/run
Ion 510™ Chip16 samples/run
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Ease of Use Through Single-Use Consumables and Easy Tracking with RFID Tags
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
Ion ReproSeq PGS Kit with Ion 520 Chips (24 samples/run)
Ion ReproSeq PGS Kit with Ion 530 Chips (96 samples/run)
Ion ReproSeq PGS Kit with Ion 510 Chips (16 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)*
Ion ReproSeq PGS Kit with Ion 520 Chips (24 samples/run)*
Ion ReproSeq PGS Kit with Ion 530 Chips (96 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
For Research Use Only. Not for use in diagnostic procedures.
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
For Research Use Only. Not for use in diagnostic procedures.
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.