Computational protocol: Evaluation of Two Highly-Multiplexed Custom Panels for Massively Parallel Semiconductor Sequencing on Paraffin DNA

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Protocol publication

[…] Custom panels targeting genomic regions and genes previously implicated in TNBC (TNBC-panel [T-panel]) and frequently altered in all breast cancer subtypes, (BREAST panel [B-panel]) were designed according to literature [–]. The design was not specifically mutation-oriented and targeted areas in the panels included intron-exon sequences in genes with reported numerical / structural alterations. The T-panel covered a total genomic sequence of ~21Kb with 286 amplicons in 43 genes; in comparison, the B-panel covered ~35Kb with 373 amplicons in 60 genes. The two panels shared 83 common amplicons with the same manufacturing amplicon ID and with identical amplification primer sequences and design region coordinates. Another 95 amplicons with different ID, primer sequences and design start-stop coordinates targeted common DNA regions (±10nts) in the two panels (shifted amplicons). An overview of overlapping targets between the two panels is shown in . Detailed sequence data and panel characteristics are shown in .Panel design was based on the GRCh37 (hg19) genomic reference. Briefly, genomic coordinates for the selected targets were exported from the UCSC Genome Browser, checked for specificity, and submitted to the Ampliseq pipeline through www.ampliseq.com. (Life Technologies / Ion Torrent, Paisley, UK). Amplicon design was adapted for FFPE samples (amplicon length of up to 150bp) and primers were delivered in two pre-mixed pools. Returned primer and amplicon designs were separately evaluated for specificity using the NCBI BLAST tool. [...] For library construction, 10ng DNA per sample were used as starting material, as per manufacturers instructions. Multiplex PCR was performed using the Ampliseq primer pools with the Ampliseq Library Kit v.2.0 and Ion Xpress barcodes, according to the manufacturer’s instructions (Life Technologies / Ion Torrent, Carlsbad, CA). Library concentration was again normalized to 15ng/ml corresponding to 100pM using Qubit HS DNA kit (Thermo Scientific, Waltham, MA). Clonal template amplification was performed on the Ion Torrent OneTouch-2 instrument followed by enrichment for template Ion Sphere Particles on a One-Touch-ES station. Templating was performed using the Ion PI template OT2-200 Kit, and sequencing was performed on an Ion Proton using PI chips (Ion PI Sequencing 200 Kit v2), with multiplexing up to 96 samples. Run metrics (mapped and on target reads, mean sample read depth, and uniformity) were evaluated in each case (shown for the FFPE tested series in Tables A—G in ).For data retrieval, base calling was performed on the Torrent Server using Torrent Suite v 3.6.2 and v 4.0. Briefly, raw data were transferred to Torrent Server and following signal processing, basecalling is performed and unmapped BAM files are generated. Filtered reads are aligned to the hg19 reference using the TMAP mapper. The TMAP integrates 3 popular alignment algorithms, BWA-short [] (<150bp), BWA-long [] (≥150bp), SSAHA [] (≥150bp), and Super-maximal Exact Matching [], while it is specifically designed for Ion Torrent products, having a principal error model relating to long homopolymer misscalls mostly resulting in insertion or deletion errors during alignment. After alignment, variant calling was performed with the embedded Variant Caller pipeline (TVC) under high stringency parameters for germline and somatic variant detection. TVC operates on a FreeBayes approach, with minor modifications to allow for Ion Torrent specific error modeling. Reads were visualized on Broad Institute Integrated Genome Viewer for integrity and target alignment. Variants generated from the TVC were uploaded to the Ion Reporter v.4 cloud except for the 44 cases from group (b) and the TN samples in group (c) that had been analyzed with v.1.6 of the same software for further annotation regarding functional effect, presence in dbSNP, COSMIC, ClinVar as well as functional classification based on SIFT, Polyphen and Grantham score. […]

Pipeline specifications

Software tools BLASTN, BWA, SSAHA, FreeBayes, PolyPhen
Databases UCSC Genome Browser
Application Genome data visualization
Diseases Breast Neoplasms, Neoplasms