Nanopanel2 calls phased low-frequency variants in Nanopore panel sequencing data

Abstract

Clinical decision making is increasingly guided by accurate and recurrent determination of presence and frequency of (somatic) variants and their haplotype through panel sequencing of disease-relevant genomic regions. Haplotype calling (phasing), however, is difficult and error prone unless variants are located on the same read which limits the ability of short-read sequencing to detect, e.g., co-occurrence of drug-resistance variants. Long-read panel sequencing enables direct phasing of amplicon variants besides having multiple other benefits, however, high error rates of current technologies prevented their applicability in the past. We have developed nanopanel2 (np2), a variant caller for Nanopore panel sequencing data. Np2 works directly on base-called FAST5 files and uses allele probability distributions and several other filters to robustly separate true from false positive calls. It effectively calls SNVs and INDELs with variant allele frequencies (VAF) as low as 1% and 5% respectively and produces only few low-frequency false-positive calls. Haplotype compositions are then determined by direct phasing. Np2 is the first somatic variant caller for Nanopore data, enabling accurate, fast (turnaround <48h) and cheap (sequencing costs ~10$/sample) diagnostic workflows.

The application of Nanopore sequencing for variant calling on the human mitochondrial DNA

“…While variant calling on long reads is still scarce, some studies reported at least comparable performance between them and conventional short reads-based pipelines (Orsini et al, 2018;Alkanaq et al, 2019;Greig et al, 2019;Wood et al, 2019;Magdy et al, 2020). Furthermore, new instruments for specific purposes have been devised recently, such as Nanopanel2 for identifying low-frequency somatic polymorphisms (Popitsch et al, 2020) or NanoVar for characterizing structural variations in low-depth sequencing data (Tham et al, 2020). However, in the studies mentioned, a single instrument was used, namely, PacBio's SMRTtools (Alkanaq et al, 2019), Nanopolish (Orsini et al, 2018;Greig et al, 2019;Magdy et al, 2020), or Varscan (Wood et al, 2019); thus, virtually no guidelines for choosing an optimal caller have been proposed.…”

“…At the same time, long reads have found application in revealing structural variants (Aganezov et al, 2020), phasing analysis with haplotype reconstruction (Maestri et al, 2020;Popitsch et al, 2020), and the nanoporebased assay for analyzing leukemic samples (Orsini et al, 2018;Cumbo et al, 2019). Nevertheless, reports on long reads' usage with regard to mitochondrial genetics are still scarce.…”