Prevalence of BRCA homopolymeric indels in an ION Torrent-based tumour-to-germline testing workflow in high-grade ovarian carcinoma

Abstract

Tumour DNA sequencing is essential for precision medicine since it guides therapeutic decisions but also fosters the identification of patients who may benefit from germline testing. Notwithstanding, the tumour-to-germline testing workflow presents a few caveats. The low sensitivity for indels at loci with sequences of identical bases (homopolymers) of ion semiconductor-based sequencing techniques represents a well-known limitation, but the prevalence of indels overlooked by these techniques in high-risk populations has not been investigated. In our study, we addressed this issue at the homopolymeric regions of BRCA1/2 in a retrospectively selected cohort of 157 patients affected with high-grade ovarian cancer and negative at tumour testing by ION Torrent sequencing. Variant allele frequency (VAF) of indels at each of the 29 investigated homopolymers was systematically revised with the IGV software. Thresholds to discriminate putative germline variants were defined by scaling the VAF to a normal distribution and calculating the outliers that exceeded the mean + 3 median-adjusted deviations of a control population. Sanger sequencing of the outliers confirmed the occurrence of only one of the five putative indels in both tumour and blood from a patient with a family history of breast cancer. Our results indicated that the prevalence of homopolymeric indels overlooked by ion semiconductor techniques is seemingly low. A careful evaluation of clinical and family history data would further help minimise this technique-bound limitation, highlighting cases in which a deeper look at these regions would be recommended.

Benchmarking and improving the performance of variant-calling pipelines with RecallME

“…Currently, the standard software for pipeline benchmarking is hap.py ( Krusche et al 2019 ) although its “simpler” version som.py is often used across several indications (both for germline and somatic VC) as it simply compares the presence of specific sequences at given positions between the ground truth and query callsets without attempting to match haplotype, which is in general problematic in cancer somatic sequencing. Som.py provides a workflow for variant harmonization and generates accuracy metrics, but suffers from specific limitations: (i) it does not provide information on quality parameters of variants identified as FN and FP; (ii) it has not been widely implemented on other VC pipelines, like Ion Torrent-based (ION) sequencing, common in clinical settings ( Azzollini et al 2023 , Ricci et al 2023 , Schnidrig et al 2023 ), for which the dominant VC algorithm is the Torrent Variant Caller (TVC). ION technology shows heterogeneity in variant annotation and some limitations in detecting insertion and deletion variants (INDELs) that may lead to incorrect estimates of the actual analytical performance ( Loman et al 2012 , Laehnemann et al 2016 , Marine et al 2020 ).…”