Assembly-based methods to detect germline structural variation using long reads

Division Of Basic Sciences - Nci

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Abstract

Long-read sequencing technologies substantially overcome the limitations of short-reads but have to date been a combination of too expensive, not scalable enough, or too noisy to be considered a feasible replacement at scale. Here we develop an efficient and scalable wet lab and computational protocol for nanopore long-read sequencing that seeks to provide a true alternative to short-reads for whole genome sequencing. We applied this protocol to cell lines and brain tissue samples as part of a pilot project for the Centers for Alzheimer's and Related Dementias (CARD). Using a single PromethIon flow cell we can detect SNPs with sensitivity/specificity better than Illumina short-read sequencing (standard for large scale genomic projects), discover structural variants (SVs) comparably to state of the art long-read based de novo assembly methods involving Pacific Biosciences HiFi sequencing and trio information, but at a much lower cost and far greater throughput, and combine and phase small and SV variants at megabase scales. The protocol also produces highly accurate, haplotype specific methylation calls. This protocol uses a de novo assembly-based framework for structural valiant discovery that improves over reference-based methods. We provide the protocol and software as open source pipelines for generating variant calls and assemblies.