Ira Deveson, the leader of the Genomic Technologies research group, is currently focusing on applying and developing long-read sequencing technologies in a diverse set of research areas. And what better place to start, than with adaptive sampling.
Requiring no special library preparation, adaptive sampling allows you to selectively target DNA sequences on-flow cell, leading to enrichment of what you want to see and depletion of what you don't, all through software. With this technique in hand, he and his group set about testing its potential for genotyping of short tandem repeat (STR) expansions - these are ~2-12 bp sequence motifs that are repeated multiple times in succession, making up ~7% of the human genome, which vary wildly in length between individuals. The variation in length can be healthy, but some expanded repeats can also act as pathogenic mutations. Many of the diseases caused by this are neurological and include Hungtington's Disease, Fragile X Syndrome, and ALS. At least 37 different genes with problematic repeat expansions are currently known to cause more than 40 neurological disorders.
Clinical diagnosis of repeat expansion disorders currently relies on labour-intensive techniques such as Southern blots, with each assay testing for only a single gene. Given the number of disorders associated with repeat expansions across many genes, which present with overlapping symptoms, clinicians currently have to make their best guess at the correct test to order which can result in a long wait for a diagnosis. Nanopore sequencing could in the future solve this problem in one step, by instead reading STRs end-end in single long reads, whilst adaptive sampling could negate the need to perform more costly whole-genome sequencing. So they decided to test this out, in conjunction with adaptive sampling, for parallel testing of all disease-associated STR genes in a single nanopore assay (this has flavours of the cancer gene panel approach taken by Phill James in the Nanopore Apps update: see Day 2 write up). Their chosen implementation of adaptive sampling was through ReadFish.
Their experiment began by cataloging all known neurological-disease-implicated STR genes and their flanking regions, which formed the targets for enrichment via adaptive sampling. An example of this being successfully implemented was shown by Ira from the HTT gene, displaying clear enrichment across the gene and lower read depth from the adjacent regions. They saw a depth of coverage of 25-35x for their targets from a single MinION Flow Cell - similar to that produced with whole-genome sequencing on a PromethION flow cell.
With the HTT gene as a focus, Ira and his team performed phasing and assembled the consensus repeat sequence for each parent. An example of this was given where one parent had a healthy copy with 18 CAG repeats, while the other had a pathogenic allele with 64 copies. In the 12 samples they tested, the adaptive sampling results correctly matched the healthy or diseased phenotypes from the clinical research samples. In fact, the STR copy numbers identified by the clinical tests were almost identical to those from nanopore sequencing, demonstrating that sequencing was at least as precise as the current, established methodologies. Some genes such as FMR1 and RFC1 cannot rely on repeat length for the identification of pathogenic variants however.
Turning attention to epigenetic modifications, these can conveniently be identified alongside nanopore sequencing. Methylated regions which are believed to be pathogenic were identified in the FMR1 region, as an example, which would lead to the silencing of this region and just so happens to be considered the main mechanism of pathogenicity in Fragile X syndrome. Ira stressed that characterizing this feature could not be done easily with any other technology. Similar real-world beneficial results were reported for the RFC1 gene, in which STR expansions are the leading cause of a debilitating ataxia, CANVAS, and nanopore sequencing was able to succinctly identify all 3 features of the pathogenic variant which currently require multiple molecular tests.
Further benefits of nanopore sequencing in these applications rely on the ability to assess several genes simultaneously, rather than one test at a time as is currently the way. This could allow for a greater understanding of the true genetic diversity at these repeat sites, which are currently poorly understood through the use of traditional sequencing technology. Where currently, the limited resolution of these genes means that the boundary between what is considered a pathogenic or non-pathogenic variant may not be clearly defined, Ira emphasized the future potential of adaptive sampling and nanopore sequencing to untangle this complex picture.
Негізгі бет Ғылым және технология Ira Deveson: Towards comprehensive genetic diagnosis of repeat expansion disorders
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