Nanopore Community Meeting 2020 collection

Speaker abstracts:

Danny E. Miller, University of Washington, USA

Targeted long-read sequencing clarifies complex genetic results and identifies missing variants

Despite advances in clinical genetic testing, many patients with suspected genetic diseases remain undiagnosed. Sometimes, testing reveals complex structural changes that are difficult to evaluate using standard clinical methods. Other times, a single variant is identified in suspected recessive disorders, or no variants at all in suspected dominant or X-linked disorders. Thus, there is a need for better tools to more fully understand cases with suspected but undiagnosed genetic diagnoses. We tested whether targeted long-read sequencing using Read Until on the Oxford Nanopore platform could be used to evaluate cases such as these. Using Read Until, we identified known pathogenic structural variants, resolved complex structural changes, and identified missing disease-causing variants in patient DNA.

Michael Dean, National Cancer Institute, USA

Analysis of human papillomavirus integration sites in cervical cancer using long-read sequencing technology

Human papillomavirus (HPV) infection is the cause of 95% of cervical cancers. We performed whole-genome long-read sequencing of the SiHa and CaSki cervical cancer cell lines covering the sequence of integrated HPV16 genomes and flanking DNA, and identified HPV concatemers too large to resolve with short-read technology. We identified 42 HPV integration sites at 39 loci not previously observed. Long-read RNA sequencing of HPV provided details on the complex HPV/human junction transcripts, and we used CRISPR cutting to target sequencing to specific loci. We plan to apply this technology to the study of cervical tumors collected in Guatemala.

Ariel Gershman, Johns Hopkins University, USA

Into the unknown: the epigenetics of repetitive DNA

In segmental duplications. While these large repetitive regions of the genome remain largely unexplored, there is evidence that epigenetic control of repeats contributes strongly to genome stability and disease. Previous attempts to study epigenetics in centromeric, pericentromeric, telomeric, and macrosatellite regions have been precluded by the difficulty in mapping conventional NGS data to large repetitive arrays. Ultra-long (>100kb) reads allow us to accurately map nanopore reads to these regions and investigate the epigenome of previously unassembled and unannotated sequences. We investigated the methylome of the centromeric higher order repeat (HOR) arrays, and observed a distinctive pattern of hyper and hypomethylation. Furthermore, we can use allele-specific methylation alone to phase microsatellite repeats, which we have demonstrated on the DXZ4 region of the X chromosome. There we found two clusters of reads which we attribute to either the active or inactive X allele. Our demonstrated ability to probe the epigenome of these repetitive areas will allow us to gain a greater understanding of their regulation and significance.

Mikhail Kolmogorov, University of California, San Diego, USA

Flye and metaFlye: algorithms for long-read de novo assembly using repeat graphs

In this talk, I will cover the algorithmic aspects of de novo genome and metagenome assembly from long reads. I will highlight our Flye assembler that uses repeat graphs to generate accurate and contigouos assemblies of various genomes. I will also present our new metagenomic assembler metaFlye, which addresses important long-read metagenomic assembly challenges, such as uneven bacterial composition and intra-species heterogeneity. Using metaFlye, we were able to recover complete or nearly-complete bacterial genome from complex environmental samples, such as human gut or cow rumen. We also showed that long-read assembly of human microbiomes enables the discovery of full-length biosynthetic gene clusters that encode biomedically important natural products.