Simultaneous detection of BRCA mutations and large genomic rearrangements in germline DNA and FFPE tumor samples

Abstract

The development of breast and ovarian cancer is strongly connected to the inactivation of the BRCA1 and BRCA2 genes by different germline and somatic alterations, and their diagnosis has great significance in targeted tumor therapy, since recently approved PARP inhibitors show high efficiency in the treatment of BRCA-deficient tumors. This raises the need for new diagnostic methods that are capable of performing an integrative mutation analysis of the BRCA genes not only from germline DNA but also from formalin-fixed and paraffin-embedded (FFPE) tumor samples. Here we describe the development of such a methodology based on next-generation sequencing and a new bioinformatics software for data analysis. The diagnostic method was initially developed on an Illumina MiSeq NGS platform using germline-mutated stem cell lines and then adapted for the Ion Torrent PGM NGS platform as well. We also investigated the usability of NGS coverage data for the detection of copy number variations and exon deletions as a replacement of the conventional MLPA technique. Finally, we tested the developed workflow on FFPE samples from breast and ovarian cancer patients. Our method meets the sensitivity and specificity requirements for the genetic diagnosis of breast and ovarian cancers both from germline and FFPE samples.

Benchmarking of Amplicon-Based Next-Generation Sequencing Panels Combined with Bioinformatics Solutions for Germline BRCA1 and BRCA2 Alteration Detection

“…24e26 FP and FN concerns have been mainly attributed to the PGM sequencing chemistry. 24,33,34 Currently, the consensus NGS technique for the detection of CNVs is the use of capturebased methods. 12 Because CNVs account for approximately 7% of all inherited BRCA alterations, 35 a workflow that allows their accurate detection at the same time as SNVs and indels is essential.…”

Investigating target‐site resistance mechanism to the PPO‐inhibiting herbicide fomesafen in waterhemp and interspecific hybridization of Amaranthus species using next generation sequencing

“…Illumina MiSeq is performed on a bench‐top device and is designed for speed and simplicity for targeted resequencing, for example PCR amplicons and small‐genome sequencing such as microorganisms, mitochondrial, and chloroplast genomes. Illumina MiSeq amplicon sequencing is a dominant platform in the study of microbial ecology and genotyping of disease related genes …”

“…Illumina MiSeq amplicon sequencing is a dominant platform in the study of microbial ecology 35,36 and genotyping of disease related genes. 34,[37][38][39] In this study, the R128 codon of PPX2 was screened in 146 PPO-R waterhemp populations from five Midwest states of the USA, including Missouri (MO), Indiana (IN), Illinois (IL), Iowa (IA) and Minnesota (MN). Illumina MiSeq amplicon sequencing was performed to access R128 codon information of pooled samples in each population.…”

A BRCA1 és BRCA2 gének tumorevolúcióban szerepet játszó mutációinak vizsgálata

“…A diagnosztikai folyamat beállítására és optimalizálására 24 különböző, ismert Torrent esetében kiegészülve a különböző variáns-szűrő alkalmazásokkal a szekvenálás minőségének in silico javítása érdekében [117].…”

“…Ennek magyarázata ebben az esetben is az allélvesztés (LOH) lehet, amely a BRCA tumorevolúció egyik fontos lépéseként a csíravonal-mutációt hordozó ováriumtumorokban is gyakori jelenség [62,134]. Ezáltal az addig heterozigóta Illumina szekvenáló platform használata mellett pedig teljesíti a diagnosztikai módszerek pontosságára és megbízhatóságára vonatkozó klinikai elvárásokat is [117].…”