ColoSeq Provides Comprehensive Lynch and Polyposis Syndrome Mutational Analysis Using Massively Parallel Sequencing

Abstract

Lynch syndrome (hereditary nonpolyposis colon cancer) and adenomatous polyposis syndromes frequently have overlapping clinical features. Current approaches for molecular genetic testing are often stepwise, taking a best-candidate gene approach with testing of additional genes if initial results are negative. We report a comprehensive assay called ColoSeq that detects all classes of mutations in Lynch and polyposis syndrome genes using targeted capture and massively parallel next-generation sequencing on the Illumina HiSeq2000 instrument. In blinded specimens and colon cancer cell lines with defined mutations, ColoSeq correctly identified 28/28 (100%) pathogenic mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH, including single nucleotide variants (SNVs), small insertions and deletions, and large copy number variants. There was 100% reproducibility of detection mutation between independent runs. The assay correctly identified 222 of 224 heterozygous SNVs (99.4%) in HapMap samples, demonstrating high sensitivity of calling all variants across each captured gene. Average coverage was greater than 320 reads per base pair when the maximum of 96 index samples with barcodes were pooled. In a specificity study of 19 control patients without cancer from different ethnic backgrounds, we did not find any pathogenic mutations but detected two variants of uncertain significance. ColoSeq offers a powerful, cost-effective means of genetic testing for Lynch and polyposis syndromes that eliminates the need for stepwise testing and multiple follow-up clinical visits.

Validation of a Next-Generation Sequencing Assay for Clinical Molecular Oncology

“…The hybridized product is amplified (5) and sequenced on an Illumina HiSeq 2000 or Illumina MiSeq instrument (6). Paired-end reads are aligned to the genome (7), PCR duplicates are removed (8), and variant calls are made (9). Variants are annotated and classified by our internally developed CGW application, using publicly available and proprietary databases, and the case is reviewed and interpreted by a clinical genomicist for sign-out in CGW (10).…”

A mosaic PTEN mutation causing Cowden syndrome identified by deep sequencing

“…Brief report been described previously. 7 Briefly, DNA was sonicated on a Covaris S2 instrument (Covaris, Woburn, MA) to a peak of 200 bp, Illumina paired-end adapters (Illumina, San Diego, CA) were ligated, and adapter-ligated library was PCR amplified for five cycles with Illumina primers 1.0 and 2.0 (Illumina). Individual paired-end libraries were hybridized to a custom design of complementary RNA biotinylated oligonucleotides targeting 1.1 Mb of DNA in 30 genomic regions including the entire PTEN gene and flanking regions.…”

“…6 We recently reported a validation study demonstrating the accuracy and feasibility of solution-based targeted capture and next-generation sequencing for genes that are implicated in Lynch and polyposis syndromes. 7 Here, we describe the application of this deep sequencing approach to identify a low-level mosaic PTEN mutation causing Cowden syndrome. Of note, this mutation was undetectable in DNA extracted from blood using traditional sequencing methods, suggesting the possibility of underrecognition of somatic PTEN mosaicism as a cause of Cowden syndrome.…”

The growing complexity of the intestinal polyposis syndromes

“…Targeted testing for multiple genes can now be done at relatively low costs and in short time. Using such targeted panels or by exome sequencing, all CRC/ polyposis genes, or specific combinations thereof, can be evaluated simultaneously with rapid turnaround times [Pritchard et al, 2012;Tanskanen et al, 2013]. In addition, mutations located in noncoding regions that are usually not covered by traditional techniques can be detected, thus increasing sensitivity.…”