Prepare Library | Sequence | Analyze Data Cumulative target size Variant types Amplicon size No. of amplicons Highlights• Relevant gene content Target all exonic regions and flanking intronic sequences of BRCA1and BRCA2 • Fast, streamlined workflow Prepare sequencing-ready libraries in a single day using as little as 1 ng high-quality DNA or 10 ng DNAfrom FFPE tissue • Accurate data Detect germline and somatic mutations down to 5% frequency using local orcloud-based analysis The AmpliSeq for Illumina BRCA Panel is a targeted resequencing assay designed for detecting somatic and germline mutations across all exonic regions and the flanking intronic sequences of BRCA1 and BRCA2(Table 1). BRCA1 and BRCA2are human tumorsuppressorgenesthat, when carrying specific mutations, have been implicated in an increased riskfor breast and ovarian cancers.1 Understanding BRCA status within the tumor may be a factor when researching potential therapies.2 To assist with quick and accurate assessment of genomic variation within BRCA1 and BRCA2, Illumina offers the AmpliSeq for Illumina BRCA Panel. The BRCA panel is part of a streamlined workflow that includes PCR-based library preparation, Illumina sequencing by synthesis (SBS) chemistry and next-generation sequencing (NGS) technology, and automated analysis. Requiring as little as 1 ng high-quality DNA per pool, the two-pool panel can be used with low-quality and low-quantity samples, including formalin-fixed, paraffin-embedded (FFPE) tissues. The AmpliSeq for Illumina BRCA Panel is part of a DNA-to-variant solution that offers streamlined content, easy-to-perform library preparation, push-button sequencing systems, and simplified data analysis. Library preparation follows a straightforward, PCR-based protocol that can be completed in as little as 5 hours, with < 1.5 hours hands-on time. Resulting libraries can be normalized, pooled, and then loaded on to a flow cell for sequencing. Prepared libraries are sequenced using proven SBS chemistry on any compatible Illumina sequencing systemthe (Table 2). Resulting data can be analyzed locally with Local Run Manageror easily streamed into BaseSpace™ Sequence Hub. Local Run Managerand BaseSpace Sequence Hub can access the DNA Amplicon analysis workflow to perform alignment and variant calling. BaseSpace Sequence Hub provides access to BaseSpace Variant Interpreter, which assists with turning variant call data into annotated results. Table 1: AmpliSeq for Illumina BRCA Panel at a glance Parameter Specification All exonic regionsofthe BRCA1 and Targets BRCA2tumor suppressor genes and flanking intronic sequences SNVs, indelsa 98 bp on average 265 Supported sample types FFPE tissue, blood Germline-, percent targets recommended throughput Somatic, percent targets covered recommended throughput Coverage uniformity (percent of targets with >0.2x mean >95% Percent on-target aligned reads > 90% Total assay timeb 5 hours Hands-on time <1.5 hours DNA-to-data time 2.5 days a. SNVs, single nucleotide variations; indels, insertions/deletions b. Time represents library preparation only and does not include library quantification, normalization, or pooling. Data on file at Illumina, Inc. 2017 Table 2: Illumina sequencing systems recommended for use with the AmpliSeq for Illumina BRCA Panel No. of Samples per Run For investigating somatic mutations iSeq™ 100 System 12 17 hours MiniSeq™ System (mid output) 24 17 hours MiniSeq System (high output) 80 24 hours MiSeq System (v2 chemistry Nano) 3 17 hours MiSeq System (v2 chemistry Micro) 12 19 hours MiSeq System (v2 chemistry) 48 24 hours MiSeq System (v3 chemistry) 80 32 hours For investigating germline mutations iSeq 100 System 96 17 hours MiSeq System (v2 chemistry Nano) 32 17 hours MiSeq System (v2 chemistry Micro) 96 19 hours A maximum of 96 samples can be multiplexed. For Research Use Only. Not for use in diagnostic procedures.

Prepare Library | Sequence | Analyze Data Call Rate Known Variant Learn more about AmpliSeq for Illumina Informatics Solutions > To demonstrate assay capabilities, Horizon Discovery (HD) and breast tissue (tumor/normal pair) samples were evaluated using the AmpliSeq for Illumina BRCA Panel on the MiSeq™ System. Results showed high coverage uniformity and on-target percentage of aligned reads, even with varying sample quality (Figure 1). In addition, two HD samples of varying quality were evaluated for variant calling accuracy. Data showed high concordance between expected and detected SNVs (Table...