MATERIALS AND METHODS – continued The Oncomine™ BRCA Research Assay was designed to cover 100% of all 23 exons of BRCA1 and 27 exons of BRCA2 with 263 different amplicons (targeted regions). We have implemented a comprehensive bioinformatics algorithm that detects LRs at high sensitivity, even in the absence of control sample(s).

Qi, Rongsu., Scafe, Charles., Nistala, Goutam., Bee, G., Garg, N., Manivannan, M., Broomer, A., Mittal, V., Williams, P., Brinza, D., Hyland, F., Bishop, J., Sadis, S., Passkiewicz, B., Sherlock, J. Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080

RESULTS We implemented additional sample QC step to detect samples with large overall amplicon coverage variations.

Table 1. Performance on simulation data.

Simulation of exon deletions: Of all exons of all samples, pick randomly 10% or 20% of exons, divide counts for all the amplicons of that exon by 2.

Table 2. Performance on blood samples.

100% exon coverage across both BRCA 1 and 2 genes, with high uniformity and read counts across all exons, allowing for over 99% confidence of detecting 5% somatic variant.

A total of 10 cell line samples purchased from Coriell were tested. From all exons of all samples, 10% or 20% of exons were selected randomly to simulate heterozygous deletion. For exon deletion simulation, the raw counts of all amplicons from that exon were divided by 2. Performance was measured by per exon per sample.

Figure 3. Visualization plots showing large deletions, single exon deletion, and multiple exon deletion from blood samples.

Boxplot of normalized count from all amplicons of each exon after re-normalization.

Boxplot of normalized count from all amplicons of each exon before and after re-normalization.

ABSTRACT We have developed an amplicon-based NGS approach for FFPE samples that can detect SNVs, small mutations and LRs simultaneously. We have implemented a comprehensive bioinformatics algorithm that detects LRs at high sensitivity, even in the absence of control sample(s). This significantly reduces the cost and labor for BRCA1/2 genetic analyses. INTRODUCTION Germline and somatic mutations in the BRCA1 and BRCA2 genes are highly involved in hereditary and non-hereditary breast and ovarian cancers. A test that detects these mutations from relevant FFPE samples is tremendously. Large rearrangements (LRs) represent a small, yet important portion of BRCA1/2 mutations, in addition to single nucleotide mutations and small insertion/deletions. The sizes of LRs make them difficult to detect using traditional sequencing approaches thereby requiring additional tests such as multiplex ligation dependent probe amplification (MLPA). Recently, several reports have shown feasibility using amplicon-based massively parallel sequencing methods to detect LRs simultaneous to small mutations. However, these tests were either not designed for use with FFPE samples, or lack data analysis methods optimized for such an application and therefore fail to achieve the necessary sensitivity. MATERIALS AND METHODS Ion AmpliSeq™, a targeted, multiplexed amplification technology for developing sequencing libraries, was used in combination with the Ion PGM and Ion S5 sequencing platform.

CONCLUSIONS We have developed an NGS assay and comprehensive data analysis approach capable of detecting both small mutations and LRs simultaneously from FFPE samples with high sensitivity and specificity. REFERENCES 1. Ava Kwong, Jiawei Chen, Vivian Y. Shin, John C.W. Ho, Fian B.F. Law, Chun Hang Au, Tsun-Leung Chan, Edmond S.K. Ma, James M. Ford: The importance of analysis of long-range rearrangement of BRCA1 and BRCA2 in genetic diagnosis of familial breast cancer. Cancer Genetics 2015, 208:448–454 2. Lídia Feliubadaló, Adriana Lopez-Doriga, Ester Castellsagué, Jesús del Valle, Mireia Menéndez, Eva Tornero, Eva Montes, Raquel Cuesta, Carolina Gómez, Olga Campos, Marta Pineda, Sara González, Victor Moreno, Joan Brunet, Ignacio Blanco, Eduard Serra, Gabriel Capellá and Conxi Lázaro:, Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes, European Journal of Human Genetics 2013, 21:864–870 ACKNOWLEDGEMENTS We thank our early access customers for sharing data with us. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

A next Generation Sequencing Approach to Detect Large Rearrangements in BRCA1/2 Simultaneous to Small Mutation Detection from FFPE Research Samples

Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com.

% exons with deletion TP FP TN FN MISS-

NOCALL NOCALL%NOCAL

LSensi-vit

y PPV

0 Original data 0 1 441 0 0 8 1.78% NA NA

10%

Simulation 1 43 2 400 1 1 4 0.89% 0.977 0.956

Simulation 2 45 0 395 0 0 0 0.00% 1 1

Simulation 3 44 3 395 0 1 8 1.78% 1 0.936

Simulation 4 44 3 392 0 0 1 0.23% 1 0.936

20 %

Simulation 1 82 4 347 4 2 3 0.68% 0.954 0.953

Simulation 2 88 0 353 1 1 8 1.78% 0.989 1

Simulation 3 75 4 343 9 3 9 2.05% 0.893 0.949

Simulation 4 85 1 352 1 1 1 0.23% 0.988 0.988

Sample type

Total sampl

es

Total exons TP FP TN FN MISSN

OCALL NOCA

LL Sensiti

vity PPV NOCA

LL_ Rate

QCFail_sampl

es

Site A blood 21 990 43 14 759 1 0 38 0.977 0.754 0.047 3

Site B blood 8 360 12 2 345 0 0 1 1 0.858 0.003 0

Figure 4. Normalized counts before and after renormalization. Before renormalization:

miscalled BRCA1 del, BRCA2 dup After renormalization: BRCA1 del, BRCA2 normal

Boxplot of normalized count from all amplicons of each exon.

log2

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When whole gene deletions are present, normalization by total number of reads is not sufficient. Re-normalization after detection of whole gene or large deletions can improve accuracy.

RESULTS - continued We first tested the performance of the pipeline using simulation data from cell lines. We achieved high sensitivity and PPV, and low No Call rate.

We then tested the assay with blood samples for germline exon deletions. Two early access customer sites provided us data from blood sample with known exon deletions. We detected a variety of single exon, multiple exon, and whole gene deletions.

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

Figure 3. Sample QC

Low variation, passed sample QC

Large variation, Failed sample QC

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Boxplot of normalized count from all amplicons of each exon.

Before re-normalization: detected E7-13 del and somatic whole gene deletion of BRCA1

Performance was measured by per exon per sample.

RESULTS - continued We then tested the assay with FFPE tumor and normal samples. We have observed in general high variability compared to blood and cell line samples. However, we were able to detect exon deletions previously detected by other methods, such as MLPA. Furthermore, our visualization tool aids manual examination for somatic deletions.

BRCA1 exons 23- 24 deletion lo

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BRCA1 exon 24 deletion

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BRCA2 exons 3-14 deletion

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BRCA1 Exons 2 duplication

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Before re-normalization: detected somatic deletion of BRCA1

After re-normalization: detected germline deletion of BRCA1 Exon 12

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Before re-normalization: high variability sample that failed QC. Visualization suggests BRCA1 Exon 22 deletion.

Figure 1. Exon coverage of BRCA1 and 2 genes by the Oncomine™ BRCA Research Assay

BRCA1 BRCA2