Haplotype-aware variant calling with PEPPER-Margin-DeepVariant enables high accuracy in nanopore long-reads

Abstract

Long-read sequencing has the potential to transform variant detection by reaching currently difficult-to-map regions and routinely linking together adjacent variations to enable read based phasing. Third-generation nanopore sequence data has demonstrated a long read length, but current interpretation methods for its novel pore-based signal have unique error profiles, making accurate analysis challenging. Here, we introduce a haplotype-aware variant calling pipeline PEPPER-Margin-DeepVariant that produces state-of-the-art variant calling results with nanopore data. We show that our nanopore-based method outperforms the short-read-based single nucleotide variant identification method at the whole genome-scale and produces high quality single nucleotide variants in segmental duplications and low-mappability regions where short-read based genotyping fails. We show that our pipeline can provide highly-contiguous phase blocks across the genome with nanopore reads, contiguously spanning between 85% to 92% of annotated genes across six samples. We also extend PEPPER-Margin-DeepVariant to PacBio HiFi data, providing an efficient solution with superior performance than the current WhatsHap-DeepVariant standard. Finally, we demonstrate de novo assembly polishing methods that use nanopore and PacBio HiFi reads to produce diploid assemblies with high accuracy (Q35+ nanopore-polished and Q40+ PacBio-HiFi-polished).

nPoRe:n-Polymer Realigner for improved pileup variant calling

“…The literature often categorizes sequences consisting of one repeated base as “homopolymers”, and repeated sequences of at least two bases as “tandem repeats” or “copolymers” [10, 23]. Short tandem repeats (STRs) are often defined as repeated units 2-6 bases in length, and are also known as “microsatellites” or “simple sequence repeats” (SSRs) [3].…”

“…The two current leading nanopore variant callers are Clair3 (developed by the HKUCS Bioinformatics Algorithm Lab) and PEPPER-Margin-DeepVariant (a collaboration between UCSC and Google Health, hereafter referred to as PEPPER) [23, 31]. Both tools have converged on a similar variant calling pipeline: basecalling, read alignment, pileup-based variant calling (using pileup summary statistics), read phasing, and full-alignment variant calling (using all read information).…”

“…Clair3 then split the model into a pileup caller to filter out the noise and a higher-dimensional full-alignment caller to make the more difficult decisions [31]. PEPPER examined sorting reads by haplotype and a new architecture, and DeepVariant explored numerous possible data representations for final calling [19, 23]. Orthogonally, we show that improved INDEL calling performance can be achieved through better read alignment by introducing novel gap penalties for homopolymers and tandem repeats, or “ n -polymers”.…”

“polishCLR: a Nextflow workflow for polishing PacBio CLR genome assemblies”

“…The polishCLR workflow will increase the efficiency of polishing many genomes and reduce the potential of human error in this multistep process. Despite the much-reduced error rate of PacBio HiFi and ONT reads, polishing approaches continue to be an important component of accurate genome assembly (Shafin et al, 2021). Although this pipeline was not designed to polish with ONT reads, the workflow is available on GitHub and welcomes any future contributions.…”

Nanopore quality score resolution can be reduced with little effect on downstream analysis

“…We denote by F z a quantizer that assigns the value F z (x) = z to every quality score x. In our experiments we take z = 10, which is a common threshold for filtering low quality reads (see, e.g., [13]). In addition, since repetitive patterns of bases are particularly difficult to sequence by nanopore technologies [14], we evaluate quantizers where the quantization of a quality score x in a position i of a read depends not only on x but also on the bases called in positions close to i.…”