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

• Published in: Nature methods Vol. 18; no. 11; pp. 1322 - 1332
• Main Authors: Shafin, Kishwar, Pesout, Trevor, Chang, Pi-Chuan, Nattestad, Maria, Kolesnikov, Alexey, Goel, Sidharth, Baid, Gunjan, Kolmogorov, Mikhail, Eizenga, Jordan M., Miga, Karen H., Carnevali, Paolo, Jain, Miten, Carroll, Andrew, Paten, Benedict
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.11.2021; Nature Publishing Group
• Subjects: 631/114/1305; 631/114/2785; 631/1647/794; 631/208/726/649; 631/61/212; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Diploids; DNA sequencing; Genes; Genetic research; Genetic variation; Genome, Human; Genomes; Genomics; Genotyping; Haplotypes; High-Throughput Nucleotide Sequencing - methods; Humans; Identification methods; Life Sciences; Methods; Molecular Sequence Annotation; Nanopores; Nanotechnology; Nucleotide sequencing; Nucleotides; Pipelines; Polymorphism, Single Nucleotide; Proteomics; Sequence Analysis, DNA - methods; Software; Vegetables; United States
• ISSN: 1548-7091; 1548-7105; 1548-7105
• DOI: 10.1038/s41592-021-01299-w

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 have demonstrated a long read length, but current interpretation methods for their 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% and 92% of annotated genes across six samples. We also extend PEPPER-Margin-DeepVariant to PacBio HiFi data, providing an efficient solution with superior performance over 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). The PEPPER-Margin-DeepVariant pipeline achieves highly accurate variant calling using nanopore and other long-read sequencing data.