Structural variants identified by Oxford Nanopore PromethION sequencing of the human genome

Abstract

We sequenced the genome of the Yoruban reference individual NA19240 on the long-read sequencing platform Oxford Nanopore PromethION for evaluation and benchmarking of recently published aligners and germline structural variant calling tools, as well as a comparison with the performance of structural variant calling from short-read sequencing data. The structural variant caller Sniffles after NGMLR or minimap2 alignment provides the most accurate results, but additional confidence or sensitivity can be obtained by a combination of multiple variant callers. Sensitive and fast results can be obtained by minimap2 for alignment and a combination of Sniffles and SVIM for variant identification. We describe a scalable workflow for identification, annotation, and characterization of tens of thousands of structural variants from long-read genome sequencing of an individual or population. By discussing the results of this well-characterized reference individual, we provide an approximation of what can be expected in future long-read sequencing studies aiming for structural variant identification.

Hecaton: reliably detecting copy number variation in plant genomes using short read sequencing data

“…Consistent with previous benchmarks performed with long read data De Coster et al, 2019), insertions remained difficult to reliably detect using short paired-end Illumina reads in all of our test cases, even after applying the filtering stage of Hecaton. We manually investigated alignments covering tens of false positive insertions in Figure 5: Performance of different CNV detection algorithms on short read data of the maize B73 accession.…”

“…After a sample of interest has been sequenced and the resulting sequencing data has been aligned to a reference genome, computational methods can extract various signals from the alignments to detect CNV between the sample and the reference . While long reads are better suited for detecting CNVs than short paired-end reads De Coster et al, 2019), sequencing data of plants is still commonly generated using short read sequencing platforms, due to their far lower cost.…”

Critical length in long read resequencing

“…The truth set of structural variants was based on a haplotype-specific direct comparison of the HG00733 assembly with the reference genome, resulting in the identification of 25139 variants larger than 50 bp, with a distribution comparable to earlier reports ( Figure S2) [7,11,15] . Structural variants from simulated reads were called using Sniffles and compared to the truth set to calculate the precision, recall, and F-measure ( Figure 1).…”

“…Long read sequencing leads to more continuous de novo genome assemblies and has advantages for genome resequencing in the context of structural variant (SV) discovery and variant phasing. It enables a more comprehensive detection of genome-wide structural variation, owing to their higher mappability in repetitive regions and their ability to anchor alignments to both sides of a breakpoint [7][8][9] . SVs are defined as genomic variability of at least 50 bp with a change in copy number or location and include deletions, insertions, inversions, and translocations [10] .…”

“…Long read resequencing has promising applications for genomics, as it enables direct observation of structural variants and inference of haplotypes [7,9,11] . In this work, we formally assess the impact of increasing read length on the accuracy of structural variant identification and haplotyping of SNVs.…”