®

Introduction•We have designed two amplicon-based, targeted NGS library

preparation (LP) panels for detecting single-nucleotide variants, insertions and deletions, copy number variants, and RNA fusions.• Both panels utilize a highly multiplexed, nanoliter-scale, PCR-based

enrichment method using an integrated fluidic circuit (IFC) that automates the reaction assembly with the Fluidigm Juno™ system.• Library preparation of 1–6 unique panels can be processed

simultaneously. Samples are assayed with LP panels in groups of 8, enabling up to 48 samples per run. • This workflow, coupled with relevant panel content and performance,

offers a solution for investigators requiring economical NGS testing of FFPE and blood samples.

Developing Solid Tumor and Hematological Panels for a Flexible and Automated Microfluidic Workflow That Improves Efficiency of NGS Library Preparation for Cancer Research

ASHG 2019: 1754F

T. Goralski1, M. Gonzales1, J. Perez1, J. Qin1, S. Chamnongpol1, D. Wang1, A. Lespagnol2, J. Jasper3,

J. Brockman1, G. Harris1, J. Wang1, J. Alipaz1, C. Park1, J.Geis1, G. Sun1, B. Fowler1, C. Kubu1, D. King1

Library prep workflow

High concordance among expected and observed single-nucleotide variant allele frequency in control samples

High replicate concordance for fusion detection in control samples

Detection at various input transcript copies. The limit of detection was determined with 10 ng input of Seraseq® reference material that contains fusion transcripts at known transcript abundance (copies).

Examples of average and standard deviations for expected allele frequencies of single-nucleotide variants detected using 12.5 ng of dilutions of NA12878 and NA24143 Genome in a Bottle control samples at positions >5% expected allele frequency.

Summary• The Advanta Solid Tumor and RNA Fusions NGS

Library Prep Assays provide relevant content for the

detection of somatic cancer-causing mutations.

• The LP 8.8.6 IFC enables sensitive and reproducible

detection of variants with 12.5 ng of input DNA and

10 ng of input RNA from FFPE samples.

• The LP 8.8.6 IFC supports multiple panel types on the

same processing run.

• The Juno workflow can prepare NGS libraries for up

to 48 samples on the LP 8.8.6 IFC in a single run, with

approximately 3.5 hours of hands on time.

• This workflow requires 50% less hands-on time than

others at similar throughput.

• These oncology panels, the LP IFC, and the Juno

system provide a flexible, cost-effective, and

convenient solution for investigators requiring

economical NGS testing of FFPE, fresh-frozen, and

blood samples.

Examples of single-nucleotide variant (SNV) detection in clinical research samples. Average allele frequency detection and replicate detection rates of variants classified as pathogenic in formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen (FF) samples.

Analytical performance: tissue samples—CNVs

Examples of copy number variant (CNV) detection in clinical research samples. Average copy numbers and replicate detection rates of variants classified as pathogenic in formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen (FF) samples.

A new IFC format for multipanel use: Juno LP 8.8.6 IFC

• Each LP 8.8.6 IFC has 6 partitions.• Tests up to 48 samples per IFC.• Each Advanta assay panel is comprised

of 8 assay pools.• Multiple assay pools enable generation

of overlapping amplicons without PCR artifacts.

• Each assay pool can easily support 200+ amplicons.

• Each partition processes 8 samples x 8 assay pools.

• Multiple panels can be processed on the same IFC.

Sample

Assays

1Fluidigm Corporation, South San Francisco, CA USA2Q2 Solutions, Morrisville, NC USA

Author disclosure information : 1: Fluidigm Corporation, South San Francisco, CA, USA; 2: Centre Hospitalier Universitaire de Rennes, 35033 Rennes Cedex, France, 3: Q2 Solutions® | EA Genomics, Durham, NC, USA

For Research Use Only. Not for use in diagnostic procedures.© Fluidigm Corporation. All rights reserved. Fluidigm, the Fluidigm logo, Advanta and Juno are trademarks and/or registered trademarks of Fluidigm Corporation in the United States and/or other countries. All other trademarks are the sole property of their respective owners. 10/2019

Results

Advanta Solid Tumor Panel

Advanta RNA Fusions Panel

Metric Definition SNVsIndels (<8 bp) CNVs Fusions

Lower limit of detection

Detection in at least 97% of replicates.

5% VAF

5% VAF

3.5 copies

250 fusion transcript copies

Positive predictive agreement

True positives/(true positives + false negatives) 98% 99% 98% 99%

Positive predictive value

True positives/(true positives + false positives) 99% 100% 97% N/A

SpecificityTrue negatives/(true negatives + false positives) 99% 100% 100% 99%

Analytical performance: control samples

Gene CDS MutationExpected Allele

FrequencyObserved Average Allele Frequency SD

ALK c.2603T>A 5% 5.3% 2.8%BRCA1 c.2082C>T 5% 5.8% 3.0%BRCA2 c.5744C>T 5% 5.1% 2.1%EGFR c.474C>T 5% 6.0% 4.2%FGFR3 c.417C>T 5% 5.7% 3.3%HRAS c.81T>C 10% 11.8% 3.9%JAK1 c.1590C>T 5% 5.4% 2.5%MAP2K2 c.660C>A 5% 6.5% 3.2%MET c.1944A>G 5% 6.3% 3.6%NF1 c.702G>A 10% 11.0% 3.6%NOTCH1 c.4049G>T 5% 8.7% 5.4%PALB2 c.1676A>G 5% 5.1% 2.0%PDGFRA c.1432T>C 5% 5.0% 2.1%RAD51D c.234C>T 5% 6.5% 2.6%RCA1 c.4308T>C 5% 5.7% 3.9%RET c.1296A>G 5% 6.2% 3.4%TERT c.3039C>T 5% 5.2% 2.5%

Fusion Event

Reported Input

Copies

Average Reported

Reads

Average Reported Reads per 1M

Reads Sequenced

% of Replicates with Detected Fusion Product

Total Replicates

PAX8-PPARG1 781 18,297 18,154 100% 152EML4-ALK 597 103,736 94,983 100% 152CD74-ROS1 474 64,338 60,046 100% 152NCOA4-RET 463 15,271 14,301 100% 152SLC34A-ROS1 370 24,312 21,644 98.7% 152SLC45A3-BRAF 361 41,025 36,632 100% 152ETV6-NTRK3 359 8,741 8,318 100% 152FGFR3-BAIAP2L1 353 7,282 6,932 100% 152KIF5B-RET 260 23,577 21,574 100% 152

FGFR3-TACC3 252 9,459 8,969 100% 152LMNA-NTRK1 241 513 488 96.7% 152TMPRSS2-ERG 172 6,824 6,372 100% 152

Gene CDS Mutation Tissue N Avg AF % Samples DetectedALK c.4472A>G Ovarian-FFPE 30 49.4% 100%ERBB2 c.1963A>G Colon-FF 11 42.2% 100%FGFR1 c.399C>A Lung-FFPE 30 3.0% 93%FGFR1 c.399C>A Colon-FFPE 12 2.4% 100%JAK1 c.3096G>A Ovarian-FFPE 11 24.5% 100%PDGFRA c.236G>A Lung-FFPE 12 35.4% 100%RET c.1784A>C Ovarian-FFPE 30 4.9% 93%TP53 c.817C>T Lung-FF 11 51.2% 100%

Analytical performance: tissue samples—SNVs

Gene Variant Tissue Replicates Avg SD % Samples >3.5 CopiesAKT1 Dup. Lung-FFPE 28 6.4 0.4 100%BRCA2 Dup. Colon-FFPE 10 3.8 0.7 80%RAC1 Dup. Lung-FFPE 10 6.4 0.9 100%RB1 Dup. Colon-FFPE 10 3.8 1 80%RICTOR Dup. Lung-FFPE 28 4.4 0.3 100%TERT Dup. Lung-FFPE 28 4.6 0.3 100%TYRP1 Dup. Colon-FF 10 4.1 0.5 100%RAC1 Del. Colon-FF 10 0.1 0 100%

31 genes:actionable hot spots

20 genes:full CDS

coverage

24 genes:CNV

targets

AKT1, BRAF, CTNNB1, EGFR, ERBB2, ERBB4, FGFR2, FGFR3, FOXL2, GNA11, GNAQ, GNAS, H3F3A, HRAS, IDH1, IDH2, KIT, KRAS, MAP2K1, MAP2K2, MAPK1, MAPK3, MET, MITF, NRAS, PDGFRA, PIK3CA, RAC1, ROS1, SMAD4, TYRP1

AKT1, ALK, BRCA1, BRCA2, CDKN2A, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, HRAS, KIT, MET, NRAS, PDGFRA, PIK3CA, PTEN, RAC1, RB1, RET, RICTOR, TERT, TYRP1, VHL

ALK, BRCA1, BRCA2, CDKN2A, DDR2, HIST1H3B, JAK1, JAK3, NF1, NOTCH1, PALB2, PTEN, RAD51C, RAD51D, RB1, RET, STK11, TERT, TP53, VHL

Panel content: Advanta Solid Tumor

Materials and Methods• For the Advanta™ Solid Tumor NGS Library Prep Assay, 1,508 primer

pairs were designed to the regions of interest with an average size of 155 bp. Over 1,000 amplicons were designed targeting known fusion breakpoints with an average insert size of 170 bp for the Advanta RNA Fusions NGS Library Prep Assay. Primer pairs for each panel were divided into 8 assay pools using informatic parameters that minimize generation of off-target products. • Sample genomic DNA, or cDNA from an off-IFC reverse

transcription reaction, together with sample barcodes and assay pools were dispensed into designated inlets on an IFC and placed in a Juno™ targeted DNA sequencing library preparation system for automated mixing of nanoscale reactions, thermal cycling, and amplicon harvesting. Harvested amplicons were collected from the IFC, pooled, and prepared for sequencing on an Illumina® NextSeq™. • The Advanta RNA Fusions panel was tested against synthetic

targets representing all targeted fusion events to demonstrate that each assay reliably detects the intended sequence of interest. For both panels, reference standards and subject FFPE samples were used to assess performance, including sensitivity, specificity, and limit of detection.

Panel content: Advanta RNA Fusions385 fusion events, >1,000 breakpoints

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View the completed gene list at fluidigm.com/advanta-rnafusions

Unique 3′Genes

Number of 5′ Partners

Unique 3′Genes

Number of 5′ Partners

Unique 3′Genes

Number of 5′ Partners

Unique 3′Genes

Number of 5′ Partners

ABI1 1 ERG 5 MLLT3 1 PRKACA 1ABI2 1 ESRP1 1 MLLT4 2 RAF1 3ABL1 11 ETV1 6 MLLT6 1 RARA 1

ACTN4 1 ETV4 5 MYB 1 RET 18AES 1 ETV5 1 MYC 7 RHEBL1 1AFF1 1 ETV6 2 MYO1F 1 ROS1 17AFF3 1 FAM22B 1 NCKIPSD 1 RPN1 1AFF4 1 FEV 2 NCOA1 1 RSPO2 1ALK 26 FGFR1 7 NCOA2 2 RSPO3 1

ARHGAP26 1 FGFR2 3 NFATC1 1 RUNX1 15ARHGEF12 1 FGFR3 2 NFATC2 1 RUNX1T1 1ASPSCR1 1 FLI1 1 NFIB 1 SARNP 1

ATF1 2 FOXO1 2 NOTCH1 2 SEPT2 1AXL 2 FOXO3 1 NOTCH2 6 SEPT5 1

BAIAP2L1 1 FOXO4 2 NOTCH3 2 SEPT6 1BCL2 3 FRYL 1 NOTCH4 1 SEPT9 1BCL6 3 FUS 1 NR4A3 4 SH3GL1 1BCOR 1 GABBR2 1 NRG1 1 SMARCA5 1BICC1 1 GAS7 1 NTRK1 13 SORBS2 1BRAF 29 GLI1 1 NTRK2 8 SP3 1

BTBD18 1 GLIS2 1 NTRK3 6 SSX1 2CASC5 1 GMPS 1 NUP214 1 SSX2 1

CBL 1 GPHN 1 NUTM1 2 SSX4 1CCAR2 1 HMGA2 1 OFD1 1 STAT6 1CDX1 1 IKZF1 3 PATZ1 1 SUZ12 1CREB1 1 ITPR2 1 PAX3 1 TACC1 1

CREB3L1 1 JAK2 6 PAX5 1 TACC3 1CREB3L2 1 KIAA0284 1 PAX8 1 TAL1 1CREBBP 1 KIAA1524 1 PBX1 2 TET1 1CRTC1 1 KIT 1 PCM1 1 TFE3 6

CTNNB1 1 LASP1 1 PDGFB 1 TOP3A 1DAB2IP 1 LPP 2 PDGFRA 5 USP6 2DAZL 1 MALT1 1 PDGFRB 4 WDFY2 1DDIT3 2 MAML2 2 PDS5A 1 WIF1 1EGFR 1 MAPRE1 1 PHF1 3 WT1 1

ELAVL3 1 MECOM 1 PICALM 1 YY1 1ELK4 1 MET 6 PLAG1 7 ZFYVE19 1ELL 1 MLLT1 1 POU5F1 1 ZNF384 1

EML4 1 MLLT10 1 PPARG 1 ZNF444 1EP300 1 MLLT11 1 PRCC 1 ZNF703 1EPS15 1

161 unique 3′ genes and the associated 5′ partner genes

Overview of new Advanta Solid Tumor and RNA Fusions NGS Library Prep Assays

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