Long-read sequencing reveals novel autism-linked variants that short-read sequencing misses, according to two preprints posted on medRxiv in July.<\/p>\n
\u201cThese are two very nice papers from top labs. They both make compelling cases for long-read sequencing,\u201d says Mi<\/a>chael<\/a> Gandal<\/a>, associate professor of psychiatry, genetics and pediatrics at the University of Pennsylvania Perelman School of Medicine, who did not contribute to either investigation. \u201cIt\u2019s likely this will become the new gold standard.\u201d<\/p>\n Conventional short-read sequencing typically outputs DNA fragments of about 150 base pairs in length. Computational tools then stitch these bits together to cover longer stretches of the genome, but they can struggle to assemble complex or repetitive regions. Newer long-read sequencing approaches<\/a> produce strips that are thousands of base pairs in length, offering better resolution of structural variants (SVs) and tandem repeats, although at a higher cost.<\/p>\n \n These are two very nice papers from top labs. They both make compelling cases for long-read sequencing. It\u2019s likely this will become the new gold standard. <\/p>\n \u2014 <\/p>\n \u2014 Large-scale genomic sequencing studies have uncovered hundreds of autism-linked genes<\/a>. But a large proportion of autism heritability<\/a> remains unexplained. Some of this variation may come from structural variants that short reads can\u2019t detect reliably, says Jonathan Sebat<\/a>, professor of psychiatry and cellular and molecular medicine at the University of California, San Diego and an investigator of one of the studies.<\/p>\n \u201cWe can find new types of mutations that we just can\u2019t see with short reads,\u201d he says.<\/p>\n Sebat and his colleagues aligned long reads to a reference genome to piece together the whole genomes of 243 people from 63 families that have at least one child with autism. Comparing these long-read data with conventional short-read data for the same participants, they pinpointed 15 autism-related rare de novo SVs in coding regions, 3 of which were detectable only with long reads, the study<\/a> shows.<\/p>\n Rather than aligning sequencing data to a reference genome, the team behind the other study<\/a> used long reads to piece together near-complete, bespoke genome assemblies for 189 people from 51 families that have at least one child with autism that has no known origin<\/a>. The team pinpointed three SVs that affect known autism-linked genes, as well as nine other SVs with potential functional consequences, most of which were missed by short reads.<\/p>\n Both studies report that long-read sequencing increased the number of autism-linked variants discovered by 4 to 6 percent.<\/p>\n That \u201cmight sound modest,\u201d wrote Evan Eichler<\/a>, professor of genome sciences at the University of Washington and an investigator of one of the studies, in an email to The Transmitter. But if rare gene variants act synergistically to increase autism likelihood, it will be essential to find them all. \u201cLong-read sequencing gets us there, short-read sequencing does not,\u201d he wrote.<\/p>\n \n S<\/p>\n ebat\u2019s team pinned down 44,647 non-tandem repeat SVs using long reads, 16,488 of which had been missed by a prior short-read analysis. But roughly 7,000 variants were detectable only by short reads, highlighting the value of using both technologies, Sebat says.<\/p>\n Most SVs occurred in noncoding regions of the genome, the study shows. But the 15 de novo SVs\u2014uninherited variants that are clearly linked to a child\u2019s condition\u2014were located in functional regions in the exome. Of these, three were identified only with long reads, six only with short reads and six with both methods.<\/p>\n \u201cIn some cases, long-read sequencing was finding variants in the exome,\u201d Gandal says. \u201cIt\u2019s significant because that in theory shouldn\u2019t happen.\u201d Short reads, he says, were thought to be accurate enough to resolve the 1 to 2 percent of the total genome that codes for genes. This shows that \u201cthat isn\u2019t the case,\u201d he adds.<\/p>\n One of these de novo SVs resulted from an in-frame duplication of STK33, which caused a \u201cradical\u201d expansion of the protein product\u2019s helical domain, as predicted by a protein folding analysis\u2014indicating that the variant likely has functional consequences, Sebat says. Two other variants, one de novo and the other inherited, resulted from complex rearrangements caused by a duplication event following a deletion, further sequence-level characterization revealed.<\/p>\n Long-read sequencing also enabled chromosome phasing\u2014the process of piecing together alleles on each parental chromosome\u2014and methylation analysis. Phasing the X chromosomes in 43 female participants revealed that the trinucleotide repeat length of FMR1, which is associated with fragile X syndrome, correlates with the gene\u2019s methylation levels<\/a> but is independent of X chromosome inactivation, a finding that aligns with previous studies. \u201cThis makes the case for using both short reads and long reads,\u201d Gandal says.<\/p>\n \n I<\/p>\n n the other study, Eichler and his colleagues compared each participant\u2019s genome with the human pangenome reference sequence<\/a> and data from the Human Genome Structural Variation Consortium, which enabled them to whittle their initial set of 33,548 SVs down to 46 total SVs of interest in the 87 children in their cohort.<\/p>\n Three of these de novo SVs\u2014two of which were absent in a comparable short-read analysis\u2014overlapped with known autism-linked genes. And nine de novo SVs coincided with exonic or regulatory regions, seven of which were missed by short reads.<\/p>\n Chromosomal phasing with these data revealed six people who each carry a rare autism-linked SV in both copies of a gene. None of the six variants identified through this analysis are found in the pangenome or in a control cohort of more than 14,000 people.<\/p>\n
\n Michael Gandal <\/p>\n