Ultra-long nanopore sequencing for assembly and scaffolding of sex chromosomes
Matthew Brian Couger, based at the Brigham and Women’s Hospital, USA, presented the creeping vole Microtus oregoni. Brian has applied ultra-long nanopore sequencing reads to solve a piece of ‘this real interesting biological puzzle’ that is this vole’s atypical sex chromosomes. This puzzle started in the 1960’s with the famous cytogeneticist Susumu Ohno, who proposed “Ohno’s Law” to explain the conservation of genes and sex chromosomes, including the novel sex chromosome system of the creeping vole. In this species, Ohno suggested that the females had an XO and the males had an XY karyotype; in gamete development, the X chromosome was contributed by the female (in the gamete), and males contributed either the Y chromosome or no sex chromosome at all.
Fascinated by this system, Brian’s team, led by Polly Campbell at UC Riverside, began to explore it using genomic sequencing. An initial ‘dive’ into this using short-read sequencing of male and female vole genomic DNA provided some insights but an incomplete picture. Their collaborator Scott Roy at San Francisco State University had similar results, with data suggesting a male specific X chromosome. To investigate where these Y genes might be on the X chromosome, they performed amplicon sequencing of Y genes, and discovered that these genes amplified in both sexes. This suggested that conserved Y genes were present in both sexes; and an independently segregating Y chromosome was absent in males. Y genes in female M. oregoni voles have been evolving independently for 150 million years, and Xist is expressed in males to compensate for having a second X chromosome.
The placement and order of these Y genes were however still unclear, and so the team performed assembly with long reads. Despite the primary long-read assembly being ‘quite excellent’, sex chromosome contigs were shorter than autosomal contigs, with repetitive sequences proving particularly problematic to resolve. They therefore performed nanopore sequencing with ultra-long reads to bridge these contig gaps. With a turnaround requirement of only one month to get the project finalised, Matthew wanted to highlight how this was a very real-world example of ‘how you can use really long reads in a project that’s not…taking you another 6 months to do all this work’. Using the recently released Ultra-long Sequencing Kit, they got an ‘amazing turnaround time for data’ from two PromethION Flow Cells, with an N50 of 91 kb, and a significant proportion of their reads being ultra-long (>50 kb). Brian stated how this demonstrates how rapidly really long reads can be obtained from a sample, without any optimisation, ‘because of just how rapid this platform is’.
The ultra-long nanopore data aligned extremely well to the genome, with high-quality alignment information. The data was highly accurate, allowing both single nucleotide variant calling and phasing. Brian highlighted a 410 kbp read that aligned to a region of the sex chromosome; ‘that’s a really solid contig for most peoples’ assemblies, not their actual read generation’. With these long reads, they could connect unassembled regions of the repeat-rich sex chromosomes. Ultra-long reads closed gaps in the initial assembly; these areas were critically important to the hypothesis as they were enriched in the sex chromosomes. Ultra-long read data generated superior alignments in repetitive regions. The position of genes relative to each other, both within a chromosome, and between the paternal X and maternal X chromosomes, was revealed. “Ohno’s puzzle” had therefore been resolved.