SCALABLE, MODULAR COMPONENTS AND RIGOROUS...
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SUMMARY • The QuantideX ® NGS workflow is flexible to accommodate low to high-throughput sample requirements, incorporating pre-analytical, in-process and post-analytical quality controls. • The kit includes pre-analytical sample qualification, 2-step PCR-based targeted library enrichment, library purification, library quantification and custom sequencing primers for use with the MiSeq ® system. • The Pan Cancer panel targets hotspot next generation sequencing of clinically relevant genes found throughout multiple tumor types. SCALABLE, MODULAR COMPONENTS AND RIGOROUS QUALITY CONTROL FOR NEXT GENERATION SEQUENCING IN THE CLINICAL LABORATORY Robyn Cardwell, Blaine Caughron, Jeff Houghton, Ted Markulin, Gary J Latham, and Andrew G Hadd Asuragen, Inc., Austin, TX INTRODUCTION Targeted assays using next-generation sequencing (NGS) are driving advances in cancer and genetic disease diagnosis. To ensure the accuracy of variant calls and relevance to precision medicine, NGS-based assays require additional layers of quality control and safeguards. A streamlined NGS assay enables rapid turnaround time from sample prep to patient reporting. With this goal in mind, we developed a modular and scalable reagent kit for NGS analysis of FFPE-extracted DNA that integrates rigorous pre-analytical, analytical and post- analytical quality controls and provides a fast and streamlined workflow. METHODS CONCLUSIONS • Our focus on controls and scalability addresses important considerations for the adoption and expeditious validation of NGS methods within the clinical laboratory. • Due to the modular workflow approach, alternative panels may be substituted to the same workflow for greater applicability to emerging targets of clinical utility. • The modular components and simple workflow may be easily translated to automated processes. Figure 1. The QuantideX NGS Pan Cancer Kit provides an all-in-one NGS solution. A comprehensive workflow integrating reagents, controls, and a novel bioinformatics suite for the sequencing of an oncology panel relevant to a diverse set of human cancers. Figure 2. The QuantideX DNA Assay can identify variations in sample quality and effect of inhibitors. Functional DNA is assessed using a qPCR-based method to assess functional copy number, which is integrated into downstream analysis. 15 Melanoma FFPE tumor biopsies were isolated with three different methods showing variation in yield (lower axis) and inhibition (upper axis). Percent amplifiable DNA is plotted with error bars for standard deviation of 3 independent runs. Figure 3. The QuantideX NGS Pan Cancer Panel uses a multiplexed primer pool and process controls to ensure the accuracy of library preparation and analysis. (A) The panel targets 21 genes across 46 regions of current and emerging targets of clinical significance in human cancer. (B) Each batch of NGS library preparation includes true FFPE positive control comprised of a 5% BRAF p.V600E mutation and a Multi-Variant process control containing 14 COSMIC variants of 3 different types (Indel, Substitution, Deletion) that are targeted by the panel. Figure 4. Dual-index codes with mastermix-free preparation ensures sample integrity. Index codes are formulated to include platform-specific adaptor sequences for targeted PCR products with custom adapters. Codes are provided in individual tubes, racked in an SBS format plate. Each sample during the indexing reaction is given a unique pairwise “set” of index codes. Figure 9. Reproducible and accurate variant call detection in controls and clinical samples is achieved using an efficient workflow. (A) Manual library preparation using the QuantideX NGS Pan Cancer Kit for 20 samples reduced hands-on operator time to 3.5 hours. (B) Titration of FFPE tumor DNA revealed dose-dependent detection of mutations to 5% of total NGS reads in multiple laboratories, maintaining sensitivity and positive predicted value (PPV). (C) Independent sample preparations and multiplex library pools from repeatability studies yielded reproducible variant calls and uniform coverage. (D) Controls included in the kit reliably reported targeted variants in the FFPE Positive Control and Multi-Variant Control across sites and runs. Figure 5. QuantideX Library Pure Prep’s bead purification methodology is simple, fast and robust to DNA copy number input. Total reads and reads passing filter is similar when procedure is varied between “standard” and “fast” in replicate analysis of FFPE samples and kit controls over broad range of input copies (100 to 24,000). Figure 6. Library Quant guides loading of the MiSeq using an internal single-point reference for the quantification of targeted NGS libraries using qPCR. Differences in cycle threshold (ΔCt) are used to quantify the sample library and guide the normalization of sample pooling. Figure 7. Premixed primers for QuantideX and standard Illumina libraries for efficient, controlled sample loading. Custom sequencing primer mixes and a diluent are provided in the kit for detection and loading of pooled QuantideX libraries and an Illumina PhiX sequencing control. The alignment of the PhiX sequencing control, mixed with QuantideX libraries, is consistent with expected results. Total reads are affected by total samples, number of amplicons, yield and sequencing quality. Figure 8. QuantideX NGS Reporter software is a locally installed NGS analysis solution. The QuantideX variant caller incorporates sample-specific pre-analytical QC data, thereby increasing the likelihood that precious samples can be processed by NGS. The number of false-positive calls in low-quality samples was dramatically reduced by the QuantideX NGS Reporter variant caller, improving the PPV of variant calling by 51% or more with only an 8% decrease in sensitivity, as compared to a caller without pre-analytical QC. Analytics & Reporting QuantideX NGS Reporter Push-Button Analytics & Reporting Suite QUANTIDEX NGS PAN CANCER KIT COMPONENTS Sample QC & Quantification Single-Tube Gene-Specific PCR Library Prep Sample Indexing & Sequencing Primers Library Purification & Quantification QuantideX qPCR DNA QC Assay QuantideX NGS Pan Cancer Panel QuantideX NGS Codes QuantideX NGS Library Pure Prep & Quant Quant Readout Inhibition Readout Gene-Specific PCR FFPE & Multi- Variant Control Barcode Tagging (Dual index Illumina or PGM Chemistries) Sequencing Primers Included for Post-Library Purification Bead Library Purification Sequencing* (MiSeq ® ) *not included Research Use Only – Not For Use In Diagnostic Procedures Preliminary research data. The performance characteristics of this assay have not yet been established. Presented at SLAS 2016 QuantideX DNA Assay QuantideX NGS Pan Cancer Panel QuantideX Codes QuantideX Library Pure Prep QuantideX Library Quant QuantideX Sequencing Reagents QuantideX NGS Reporter RESULTS Quant Reporter Inhibition Reporter 4-Point DNA Standard Curve FFPE or FNA DNA QuantideX ® DNA Assay Determine amplifiable DNA copies, flag any functional PCR inhibition, qualify input into enrichment EGFR BRAF KIT KRAS BRAF NRAS BRAF RET Lung Melanoma Colorectal Breast/Ovarian ABL1 Thyroid Leukemia/Lymphoma AKT1 BRAF RET MET ERBB2 FGFR1 NRAS KRAS ALK1 PIK3CA NRAS AKT1 AKT2 EGFR PIK3CA MET AKT1 AKT2 PIK3CA ERBB2 KRAS NRAS FGFR3 FLT3 JAK2 AKT1 ALK1 BRAF EGFR FGFR1 FGFR3 HRAS IDH1 IDH2 KRAS MET NRAS PDGFRA PIK3CA RET In Clinical Guidelines Emerging Therapeutic Targets Pancreatic/Gastric/Sarcomas/ Glioblastomas & Other 5’-P5-Index- 3’ Target Seq 3’ -Index-P7- 5’ P5 Index2 Index1 { QuantideX Primer Illumina Primer 700,000 600,000 500,000 400,000 300,000 200,000 100,000 Number of Reads Sample Type and Input Copy Number Comparison of Total and Passing Filter Reads by Library Pure Prep Method 0 Mean Total Reads; Fast Mean PF Reads; Fast Total Reads; Standard PF Reads; Standard Cell Line gDNA (100) Cell Line gDNA (200) Breast Cancer FFPE Colorectal Cancer FFPE Melanoma FFPE Cell line gDNA (400) FFPE Positive Control Multi-Variant Control Cell line gDNA (3000) Cell line gDNA (12000) Cell line gDNA (24000) 100 200 400 500 3000 12000 24000 PhiX Alignment Samples/Run Expected Observed 8 7.40% 7.50% 81 7.40% 8.20% 192 7.40% 6.10% Set-up QC GS PCR Library Clean Up Library Quant Norm/Pooling Operator Time: 3.5 Hours Total Library Prep Time: 11 Hours Tagging PCR 0 3 6 9 12 Time Hours DNA QC Gene- specific PCR Index Codes Library Purification Library Quant Sequencing Primers Bioinformatics Sample ID Inhibition (Cq) 1 of 3 Inhibited 2 of 3 Inhibited 3 of 3 Inhibited Isolation Method #1 #2 #3 Samples can be re-purified to qualify for NGS 40 38 36 34 32 30 10.0 7.5 5.0 2.5 0 23% CV: 2 Op, 3 runs 24% CV: 2 Op, 3 runs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 % Amplifiable DNA (Functional Cp#/total Cp) Multi-Variant Ctrl FFPE Positive Ctrl DNA QC fastq 9A 3A 9C 9B 3B 9D Method Standard Fast Bind off magnet? Yes Yes Bind incubation 4 min -- Bind magnet time 4 min 20 sec Wash off magnet? Yes No Resuspend beads during wash? Yes No Wash incubation 2 min -- Wash magnet time 2 min 20 sec Dry incubation time 2 min 1 min Elute off magnet? Yes Yes Elute incubation 4 min 1 min Elute magnet time 4 min 20 sec Total plate moves (on/off magnet) 7 3 Total incubation and magnet time 26 min 3.33 min
Transcript of SCALABLE, MODULAR COMPONENTS AND RIGOROUS...
SUMMARY• The QuantideX® NGS workflow is flexible to
accommodate low to high-throughput sample requirements, incorporating pre-analytical, in-process and post-analytical quality controls.
• The kit includes pre-analytical sample qualification, 2-step PCR-based targeted library enrichment, library purification, library quantification and custom sequencing primers for use with the MiSeq® system.
• The Pan Cancer panel targets hotspot next generation sequencing of clinically relevant genes found throughout multiple tumor types.
SCALABLE, MODULAR COMPONENTS AND RIGOROUS QUALITY CONTROL FOR NEXT GENERATION SEQUENCING IN THE CLINICAL LABORATORYRobyn Cardwell, Blaine Caughron, Jeff Houghton, Ted Markulin, Gary J Latham, and Andrew G HaddAsuragen, Inc., Austin, TX
INTRODUCTIONTargeted assays using next-generation sequencing (NGS) are driving advances in cancer and genetic disease diagnosis. To ensure the accuracy of variant calls and relevance to precision medicine, NGS-based assays require additional layers of quality control and safeguards. A streamlined NGS assay enables rapid turnaround time from sample prep to patient reporting. With this goal in mind, we developed a modular and scalable reagent kit for NGS analysis of FFPE-extracted DNA that integrates rigorous pre-analytical, analytical and post-analytical quality controls and provides a fast and streamlined workflow.
METHODS
CONCLUSIONS• Our focus on controls and scalability addresses
important considerations for the adoption and expeditious validation of NGS methods within the clinical laboratory.
• Due to the modular workflow approach, alternative panels may be substituted to the same workflow for greater applicability to emerging targets of clinical utility.
• The modular components and simple workflow may be easily translated to automated processes.
Figure 1. The QuantideX NGS Pan Cancer Kit provides an all-in-one NGS solution. A comprehensive workflow integrating reagents, controls, and a novel bioinformatics suite for the sequencing of an oncology panel relevant to a diverse set of human cancers.
Figure 2. The QuantideX DNA Assay can identify variations in sample quality and effect of inhibitors. Functional DNA is assessed using a qPCR-based method to assess functional copy number, which is integrated into downstream analysis. 15 Melanoma FFPE tumor biopsies were isolated with three different methods showing variation in yield (lower axis) and inhibition (upper axis). Percent amplifiable DNA is plotted with error bars for standard deviation of 3 independent runs.
Figure 3. The QuantideX NGS Pan Cancer Panel uses a multiplexed primer pool and process controls to ensure the accuracy of library preparation and analysis. (A) The panel targets 21 genes across 46 regions of current and emerging targets of clinical significance in human cancer. (B) Each batch of NGS library preparation includes true FFPE positive control comprised of a 5% BRAF p.V600E mutation and a Multi-Variant process control containing 14 COSMIC variants of 3 different types (Indel, Substitution, Deletion) that are targeted by the panel.
Figure 4. Dual-index codes with mastermix-free preparation ensures sample integrity. Index codes are formulated to include platform-specific adaptor sequences for targeted PCR products with custom adapters. Codes are provided in individual tubes, racked in an SBS format plate. Each sample during the indexing reaction is given a unique pairwise “set” of index codes.
Figure 9. Reproducible and accurate variant call detection in controls and clinical samples is achieved using an efficient workflow. (A) Manual library preparation using the QuantideX NGS Pan Cancer Kit for 20 samples reduced hands-on operator time to 3.5 hours. (B) Titration of FFPE tumor DNA revealed dose-dependent detection of mutations to 5% of total NGS reads in multiple laboratories, maintaining sensitivity and positive predicted value (PPV). (C) Independent sample preparations and multiplex library pools from repeatability studies yielded reproducible variant calls and uniform coverage. (D) Controls included in the kit reliably reported targeted variants in the FFPE Positive Control and Multi-Variant Control across sites and runs.
Figure 5. QuantideX Library Pure Prep’s bead purification methodology is simple, fast and robust to DNA copy number input. Total reads and reads passing filter is similar when procedure is varied between “standard” and “fast” in replicate analysis of FFPE samples and kit controls over broad range of input copies (100 to 24,000).
Figure 6. Library Quant guides loading of the MiSeq using an internal single-point reference for the quantification of targeted NGS libraries using qPCR. Differences in cycle threshold (ΔCt) are used to quantify the sample library and guide the normalization of sample pooling.
Figure 7. Premixed primers for QuantideX and standard Illumina libraries for efficient, controlled sample loading. Custom sequencing primer mixes and a diluent are provided in the kit for detection and loading of pooled QuantideX libraries and an Illumina PhiX sequencing control. The alignment of the PhiX sequencing control, mixed with QuantideX libraries, is consistent with expected results. Total reads are affected by total samples, number of amplicons, yield and sequencing quality.
Figure 8. QuantideX NGS Reporter software is a locally installed NGS analysis solution. The QuantideX variant caller incorporates sample-specific pre-analytical QC data, thereby increasing the likelihood that precious samples can be processed by NGS. The number of false-positive calls in low-quality samples was dramatically reduced by the QuantideX NGS Reporter variant caller, improving the PPV of variant calling by 51% or more with only an 8% decrease in sensitivity, as compared to a caller without pre-analytical QC.
Analytics &Reporting
QuantideXNGS Reporter
Push-ButtonAnalytics &
Reporting Suite
QUANTIDEX NGS PAN CANCER KIT COMPONENTS
Sample QC &Quantification
Single-Tube Gene-Specific
PCR Library Prep
Sample Indexing & Sequencing
Primers
Library Purification & Quantification
QuantideX qPCR DNA QC Assay
QuantideXNGS Pan
Cancer Panel
QuantideXNGS Codes
QuantideXNGS Library Pure
Prep & Quant
Quant Readout
Inhibition Readout
Gene-Specific PCR
FFPE & Multi-Variant Control
Barcode Tagging (Dual index Illumina
or PGM Chemistries)
Sequencing Primers Included for Post-Library
Purification
Bead Library Purification
Seq
uenc
ing*
(MiS
eq®)
*not included
Research Use Only – Not For Use In Diagnostic ProceduresPreliminary research data. The performance characteristics of this assay have not yet been established.
Presented at SLAS 2016
QuantideX DNA Assay
QuantideX NGS Pan Cancer Panel
QuantideX Codes
QuantideX Library Pure Prep
QuantideX Library Quant
QuantideX Sequencing Reagents
QuantideX NGS Reporter
RESULTS
Quant Reporter Inhibition Reporter
4-Point DNAStandard Curve
FFPE or FNADNA
QuantideX® DNA Assay
Determine amplifiable DNA copies,flag any functional PCR inhibition,
qualify input into enrichment
EGFR
BRAFKIT
KRASBRAFNRAS
BRAFRET
Lung Melanoma Colorectal Breast/Ovarian
ABL1
Thyroid Leukemia/Lymphoma
AKT1BRAFRETMETERBB2
FGFR1NRASKRASALK1
PIK3CANRAS
AKT1AKT2EGFRPIK3CAMET
AKT1AKT2PIK3CAERBB2
KRASNRAS
FGFR3FLT3JAK2
AKT1ALK1BRAFEGFRFGFR1
FGFR3HRASIDH1IDH2KRAS
METNRASPDGFRAPIK3CARET
In C
linic
alG
uid
elin
esE
mer
gin
gTh
erap
euti
c Ta
rget
s
Pancreatic/Gastric/Sarcomas/Glioblastomas & Other
5’-P5-Index-3’
Target Seq
3’-Index-P7-5’
P5 Index2 Index1
{QuantideX Primer Illumina Primer
700,000
600,000
500,000
400,000
300,000
200,000
100,000
Num
ber o
f Rea
ds
Sample Type and Input Copy Number
Comparison of Total and Passing Filter Reads by Library Pure Prep Method
0
Mean Total Reads; FastMean PF Reads; FastTotal Reads; StandardPF Reads; Standard
Cell Li
ne gD
NA (100
)
Cell Li
ne gD
NA (200
)
Breast
Cance
r FFPE
Colorec
tal C
ance
r FFPE
Melano
ma FFPE
Cell lin
e gDNA (4
00)
FFPE Pos
itive C
ontro
l
Multi-V
arian
t Con
trol
Cell lin
e gDNA (3
000)
Cell lin
e gDNA (1
2000
)
Cell lin
e gDNA (2
4000
)100 200 400 500 3000 12000 24000
PhiX Alignment
Samples/Run Expected Observed
8 7.40% 7.50%
81 7.40% 8.20%
192 7.40% 6.10%
Set-up QC
GS PCR
Library Clean Up
Library Quant
Norm/Pooling
Operator Time: 3.5 Hours Total Library Prep Time: 11 Hours
Tagging PCR
0
3
6
9
12
Tim
e H
our
s
DNA QCGene-
specificPCR
Index Codes Library Purification
Library Quant
Sequencing Primers
Bioinformatics
Sample ID
Inhi
bitio
n(C
q)
1 of 3 Inhibited
2 of 3 Inhibited
3 of 3 Inhibited
Isolation Method #1
#2
#3
Samples canbe re-purified toqualify for NGS 40
38
36
34
32
30
10.0
7.5
5.0
2.5
0
23% CV: 2 Op, 3 runs 24% CV: 2 Op, 3 runs
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
% A
mpl
ifiab
le D
NA
(Fun
ctio
nal C
p#/to
tal C
p)
Mul
ti-Va
riant
Ctr
l
FFPE
Pos
itive
Ctr
l
DNA QC
fastq
9A
3A
9C
9B
3B
9D
Method Standard Fast
Bind off magnet? Yes Yes
Bind incubation 4 min --
Bind magnet time 4 min 20 sec
Wash off magnet? Yes No
Resuspend beads during wash? Yes No
Wash incubation 2 min --
Wash magnet time 2 min 20 sec
Dry incubation time 2 min 1 min
Elute off magnet? Yes Yes
Elute incubation 4 min 1 min
Elute magnet time 4 min 20 sec
Total plate moves (on/off magnet) 7 3
Total incubation and magnet time 26 min 3.33 min
