Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Abstract

Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies (ONT) PromethION platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables genome-wide detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from the long-read data achieved good specificity and sensitivity.However, results of somatic SNV calling highlight the need for the development of specialized joint calling algorithms. We find the comparative performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans.

Unravelling the tumour genome: The evolutionary and clinical impacts of structural variants in tumourigenesis

“…Most of the SVs discovered using ONT and PacBio reads are novel, lending weight to the notion that a large number of SVs are not detected by short‐read approaches [52,79–81]. In addition, longer read lengths allow haplotype phasing and de novo assembly of complex SVs, crucial for the accurate interpretation of the functional impact of these events [82–85]. Taking advantage of the unique properties of both ONT and PacBio long‐read sequencing, and the development of new whole‐genome assembly methods [78,86], the Telomere‐to‐Telomere Consortium has recently assembled a complete human genome for the first time [72,87,88].…”

“…Most of the SVs discovered using ONT and PacBio reads are novel, lending weight to the notion that a large number of SVs are not detected by short-read approaches [52,[79][80][81]. In addition, longer read lengths allow haplotype phasing and de novo assembly of complex SVs, crucial for the accurate interpretation of the functional impact of these events [82][83][84][85]. Taking advantage of the unique properties of both ONT and PacBio long-read sequencing, and the development of new whole-genome assembly methods [78,86], the Telomere-to-Telomere Consortium has recently (i) chromothripsis where the genome is shattered and the pieces joined back together at random, (ii) chromoplexy where interdependent translocations and deletions are joined together in a chain, (iii) chromoanasynthesis, which is a pattern of localised amplification and short stretches of breakpoint microhomology, (iv) breakage-fusion-bridge cycles, where telomeres are lost, dicentric chromosomes form before breaking due to mitotic stress, (v) extrachromosomal DNAs (ecDNAs), where large segments of DNA form acentric circles that often contain oncogenes and regulatory regions; these ecDNAs may be reincorporated into the chromosome, as (vi) homogeneously staining regions, and (vii) aneuploidy and whole-genome duplication where some or all chromosomes have additional or double the number of copies, respectively.…”

PRINCESS: comprehensive detection of haplotype resolved SNVs, SVs, and methylation

“…Recent long-read studies often focus on either phasing of SNV, SV, or methylation detection [30,31,54]. Yet, the information of all three variant/modification types are available over long-read platforms [55].…”

Somatic mosaicism detected by genome-wide sequencing in 500 parent–child trios with suspected genetic disease: clinical and genetic counseling implications

“…SNP microarray technology is well suited for detecting genomic copy number mosaicism, as probe density is high (King et al 2017), but CMA cannot detect balanced structural variants or copy number variants smaller than 100,000 bp or so. NGS technologies can detect mosaicism for smaller copy number variants (King et al 2015(King et al , 2017Balachandran and Beck 2020), and newer research technologies like long-read sequencing show great promise for detecting mosaicism involving small, balanced, and complex structural variants as well (Fujimoto et al 2021;Roberts et al 2021).…”