Ultra-long reads from Australian reptiles
Jillian began her talk by discussing the importance of ultra-long reads, and how they formed the basis of her research. In order to express their importance in assembly she used an analogy of piecing together a jigsaw puzzle, which just like genome assembly becomes much harder with short pieces and easier with long pieces. Starting off with a broad overview, she stated that ultra-long reads result in more accurate assemblies, with fewer gaps – largely attributed to a greater propensity to include whole repetitive regions or hard to sequence areas. In the same way, ultra-long read assemblies are more likely to include structural rearrangements. Finally, Jillian explained their utility for phasing.
Jillian went on to introduce her project on using ultra-long reads to generate high-quality reference assemblies for two native Australian reptiles; the bearded dragon and the shingleback lizard. The broader aim was to create a high-quality resource for labs studying these species, as well as meet the imperative representation across Australian reptile phylogeny. In addition, Jillian expressed a more specific aim of the project was to discriminate between the highly homologous sex chromosomes. Discussing the sex chromosome system in more detail, Jillian stated that the sex of the species 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, albeit at higher egg incubation temperatures this is overridden, and both become female. Jillian said the genes and mechanisms underlying this process are not fully understood, and this is in part down to the fact that efforts to assemble and phase the highly repetitive and homologous chromosomes Z/W have been unsuccessful. To that end, Jillian is in the process of using ultra-long reads to assemble and phase chromosomes Z/W in order to better understand reptile sex determination.
Jillian then moved on to her second project, involving two highly venomous sea snakes, native to the Indian and Pacific oceans; the Shaw’s sea snake and the ornate reef sea snake. Like with the reptiles, she aimed to use ultra-long reads to produce high-quality reference genomes, with the ultimate aim of comparing genomes with their terrestrial snake relatives. Jillian explained how this would hopefully shed light on how sea snakes have adapted to live in a marine environment. One such way is to take in oxygen through their skin and expel nitrogen and carbon dioxide. Again, the genes and underlying mechanisms associated with this phenotype remain unclear.
Jillian said her work is in its infancy and they are currently working on developing the methodology. She went on to discuss her methodology for obtaining ultra-long reads, kicking off with DNA extraction and sequencing. She has been using the ultra-long sequencing products developed by Circulomics and Oxford Nanopore, and briefly touched upon the finer details of how the extraction and library preparation works. She mentioned that in ‘in my hands these protocols take about an hour to an hour and a half’ with an overnight incubation for the elution step. Jillian proceeded to dig deeper into the DNA extraction process. She expressed the convenience of reptile red blood cells containing DNA, and therefore requiring much lower inputs of blood when compared to mammalian samples. She added that there was a little variation between species for the amounts of input blood needed to get sufficient DNA, with less required for the reptiles and more required for the sea snakes. Jillian stated that all of their extractions were carried out from frozen blood, and noted that she is still able to get high-quality high-molecular weight DNA.
Jillian moved on to compare the read length for the bearded dragon and shingle back lizard using standard techniques and the Ultra-Long Sequencing Kit. The standard method, which Jillian pointed out was used prior to the availability of the ultra-long kit, used DNA spooling and an older Circulomics Nanobind kit, followed by a standard ligation library preparation (LSK109/LSK110). Jillian observed a marked increase in N50, which essentially doubled in both cases from around 35 kb to over 70 kb. On top of that, using the ultra-long kits she is now consistently seeing over a third of the reads exceeding 100 kb, with a significant number exceeding 500 kb. Jillian noted that the output and N50 was slightly less in the bearded dragon compared to the other organisms because she used a slightly earlier iteration of the kit. However, with the latest version of the kit Jillian reports getting consistently over 80 kb N50s, and an average output of around 60 Gb. Jillian moved on to show a table of her draft genome assemblies to date, which were all performed with FLYE. She pointed out she has polished the bearded dragon and shingleback lizard genomes, thereby improving the BUSCO scores, and she intends to do the same with the two sea snakes.
Jillian concluded the talk by outlining her next steps. She intends to perform another ultra-long run on each sea snake to obtain an even greater coverage, which she will subsequently polish with short-read data. She plans to use Hi-C data for each reptile in order to assemble the contigs into chromosomes. Finally, Jillian mentioned about performing cDNA sequencing in order to annotate the transcriptome. After achieving this, she hopes to be able to better elucidate the mechanisms that drive sex determination in reptiles and better understand aquatic adaptations in sea snakes.