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Shedding light on long-range interactions of the human epigenome
Studying how the genome and the epigenome interact to regulate gene transcription improves our understanding of diseases such as cancer. Roham Razaghi recently explained how his team at Johns Hopkins University, USA, wanted to examine long-range promoter-enhancer interactions and their respective methylation status within the human genome.* Promoter methylation is known to confer gene silencing, whereas unmethylated promoters are typically associated with transcriptional activity. Enhancer methylation, on the other hand, results in a more nuanced regulation of gene silencing. According to Roham, traditional methods offering simultaneous analysis of promoter-enhancer interactions and methylation status cannot do so at the single-read level, nor can they distinguish allele-specific interactions; however, Roham believed that nanopore sequencing, which combines long reads with direct detection of modified bases, “could address both of these concerns.”
Using the newly developed Ultra-Long DNA Sequencing Kit and workflow, jointly developed by Oxford Nanopore and Circulomics, the researchers performed whole-genome sequencing of a colorectal cancer cell line (HCT-116) on a single PromethION Flow Cell. A genome coverage of 40X, with an N50 of 98 kb, and reads exceeding 2 Mb were obtained. Roham emphasized the utility of ultra-long nanopore reads, saying “we can look at methylation status across a whole gene body and their regulatory elements.” Using this approach to study the CCN1 gene, which when upregulated correlates with poor prognosis in colorectal cancer, the team observed clear heterogeneity of methylation status across the gene’s three enhancers. Their results were in line with previous literature, which reports the highest activity in Enhancer 3 and it being largely responsible for CCN1 upregulation. Roham stated that he had “not been able to capture this information with other methods’ and believes it will guide the team’s refinement of promoter-enhancer loop models.
Solving Ohno’s puzzle—resolving an ancient sex chromosome system
In the 1960s, prominent cytogeneticist Susumu Ohno described an atypical sex chromosome system in the creeping vole (Microtus Oregoni). He proposed that females had an XO and males had an XY karyotype; with males contributing either the Y chromosome or no sex chromosome during gamete development. Although sparking much debate at the time, the mechanisms behind this puzzle remained unresolved for almost 60 years, until Matthew Brian Couger, Brigham and Women’s Hospital, USA, and colleagues took up the challenge.*
An initial dive into this fascinating system saw the use of short-read sequencing of male and female vole genomic DNA and pointed to a male-specific X chromosome. Intriguingly, amplicon sequencing of conserved Y chromosome genes revealed their presence in both male and female voles. No such Y chromosome genes were detected in females of closely related Microtus species, indicating that this system had been evolving independently for approximately 150 million years.
The placement and order of these Y genes was, however, still unclear, and so the team performed genome assembly with long reads. Despite the initial long-read assembly being “quite excellent”, sex chromosome contigs were shorter than autosomal contigs, likely due to the highly repetitive nature of the sex chromosomes. To resolve this, the team turned to ultra-long nanopore sequencing reads. Using the recently released Ultra-Long DNA 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).
The high-quality, ultra-long nanopore data aligned “extremely well” to the genome, and further supported accurate SNP calling and phasing. Highlighting a 410 kb read that aligned to a region of the sex chromosome, Matthew commented: “that’s a really solid contig for most peoples’ assemblies, not their actual read generation.” With these long reads, unassembled regions of the repeat-rich sex chromosomes were connected. The position of genes relative to each other, both within a chromosome, and between the paternal X and maternal X chromosomes, was revealed. And so Ohno’s puzzle was resolved, revealing a unique sex chromosome system whereby male gametes contain either a paternal X chromosome, generating male offspring, or no X chromosome, giving rise to female offspring.
Ultra-long reads from Australian reptiles
At the Garvan Institute, Australia, Jillian Hammond is also using ultra-long nanopore sequencing reads to resolve previously uncharacterized genomic regions, including the highly repetitive, highly homologous sex chromosomes observed in Australian reptiles.* Interestingly, the sex of many reptiles is determined by both genotype and the environment, in particular the temperature. Typically, a ZZ genotype becomes a male and a ZW genotype becomes a female; however, at higher egg incubation temperatures this is overridden, and both become female.
According to Jillian, over a third of the reads obtained using the Ultra-Long DNA Sequencing Kit consistently exceeded 100 kb, with a significant number exceeding 500 kb. Furthermore, read N50s doubled from around 35 kb to over 70 kb when compared to their previous workflow. Draft genome assemblies for the bearded dragon, the Shingleback lizard, and two highly venomous sea snakes have now been generated using FLYE, with the team planning to further enhance these assemblies using Hi-C data and cDNA-based gene annotation. Using the completed assembles, they aim to elucidate the mechanisms that drive sex determination and better understand aquatic adaptations in sea snakes.
* London Calling 2021 online conference, hosted by Oxford Nanopore Technologies; May 19–21, 2021.
Watch all London Calling Ultra-Long Read talks: www.nanoporetech.com/ultra-long-sequencing
