Sequencing snakes: the BABS Genomes at SBRS 2018

This month saw the first presentation of the BABS genomes outside UNSW. A poster on the genome assemblies was presented at the Sydney Bioinformatics Research Symposium 2018 (abstract below):

Pseudodiploid pseudo-long-read whole genome sequencing and assembly of Pseudonaja textilis (eastern brown snake) and Notechis scutatus (mainland tiger snake)

Richard J Edwards, Timothy G Amos, Joshua Tang, Beni Cawood, Sabrina Rispin, Daniel Enosi Tuipulotu & Paul Waters.

Click on thumbnail for full resolution PDF. If you use anything from this work, please cite:

Edwards RJ et al. Pseudodiploid pseudo-long-read whole genome sequencing and assembly of Pseudonaja textilis (eastern brown snake) and Notechis scutatus (mainland tiger snake) [version 1; not peer reviewed]. F1000Research 2018, 7:753 (poster) (doi: 10.7490/f1000research.1115550.1)

Abstract

The precipitous drop in sequencing costs over recent years has seen the bottleneck in vertebrate whole genome sequencing (WGS) shift from data generation (sequencing) to data processing (assembly and annotation). Draft genomes generated from cheap shotgun Illumina sequencing tend to be highly fragmented with many tens of thousands of short contigs or scaffolds. This can be improved by preparing multiple paired end and “mate pair” libraries with different insert sizes, but this increases the cost of both sequencing and data storage/analysis. PacBio or Oxford Nanopore long read sequencing enables massive improvements in assembly quality but tends to be prohibitively expensive for organisms with large genome sizes, such as vertebrates. 10x Genomics Chromium “linked read” sequencing offers a solution to this problem. High molecular weight molecules of DNA are barcoded prior to standard shotgun Illumina sequencing. These barcodes can then be used for pseudo-long-read assembly, with improved handling of repetitive regions. Where heterozygous variants are dense enough, haplotypes can be phased to generate a “pseudodiploid” assembly with some regions represented as two alleles. This is all for the cost of an additional library prep with no extra sequencing. But does it work?

We have sequenced two of the deadliest venomous snakes in Australia using 10x Chromium linked reads: the mainland tiger snake (Notechis scutatus) and the eastern brown snake (Pseudonaja textilis). Supernova v2 assemblies of the data generated exceptionally high quality genomes for the price, with maximum scaffolds over 50 Mb and N50 values of 5.99 Mb for the tiger snake and 14.7 Mb for the brown snake. This was reflected in BUSCO (v2.0.1 short) completeness estimates of 87.3% (tiger snake) and 90.5% (brown snake). These data will be compared to tiger snake WGS using standard paired end Illumina NovaSeq shotgun sequencing, and discussed with respect to some of the downstream opportunities and challenges provided by pseudodiploid genome assemblies. In particular, BUSCO analysis of haploid, pseudodiploid, and non-redundant genome assemblies revealed some interesting and unexpected behaviour of this widely-used tool. We also present results from GenomeR, a Shiny app (in development) for batch kmer genome size estimation (http://shiny.slimsuite.unsw.edu.au/GenomeR/).

Snake genomes and ongoing annotation are being made available through the lab Web Apollo browser and search tool (https://slimsuite.unsw.edu.au/servers/apollo.php). We welcome contact from anyone interested in getting involved with the annotation and analysis of these genomes.

Watch this blog for more details on different aspects of the analysis.

Where do our snakes come from?

The snakes we are sequencing for the BABS Genome project were kindly supplied by Nathan Dunstan at Venom Supplies as a collaborative contribution to Paul Waters and Denis O’Meally when they were at ANU. Thanks Nathan!

We have sequenced two Tiger snake parents, originally caught from the southeast of South Australia (just north of Mt Gambier) in about 2004. They were bred at Venom Supplies, and we have also sequenced one of the babies (sex unknown) born in February 2013.

The brown snake was a female from a clutch of eggs from a gravid (pregnant) female caught locally in the Barossa.

Photo Credits

Tiger Snake (left): Teneche [CC BY-SA 3.0] | Brown Snake (right): Denis O'Meally.

Linked read sequencing is go!

We already have over four billion reads and 620 GB of NovaSeq Illumina data for our three tiger snakes; next week’s BABS3291 prac will look at some of the early ABySS assemblies of one of these snakes.

Phase 2 of the sequencing is now go! 10x Chromium linked read libraries were prepared at the Ramaciotti Centre for Genomics last week for one tiger snake and one eastern brown snake. These data promise to make much easier and more intact genome assemblies. We received notification today that the samples have arrived in the KCCG sequencing laboratory at the Garvan Institute for Illumina HiSeq X (“XTen”) sequencing.

Nobody knows how well linked read sequencing, which is optimised for human genomes, will work in a snake but we look forward to finding out!

We're sequencing snakes! (But the competition's still on...)

Today was the first BABS3291 Genes, Genomes and Evolution lecture and the official opening of the new Ramaciotti Centre for Genomics labs in the shiny new E26 Bioscience South building at UNSW. This seemed like an appropriate day to reveal the chosen organisms for the BABS2017 Genome.

And the answer is... two snake species!

And not just any snakes… the Tiger Snake (left) and Brown snake (right, narrowly avoided by my postdoc Åsa and her partner!) are two of the most deadly snakes in the world. More information will follow in future posts.

Data from the BABS Genome is going to form the core of the seven-week genomics bioinformatics practical at the heart of the coursework for BABS3291. This obviously meant that we needed to start generating data before the course started. We’re still keen to find out what you would like sequenced, though, and the BABS Genome competition remains open for now. Who knows, you may help pick the next genome we do!

Photo Credits

• Tiger Snake: Teneche - CC BY-SA 3.0. Location: Banyule Flats Reserve, Melbourne, Victoria.

• Brown Snake: Patrick Dessi. Location: Booroomba Rocks, Namadgi National Park, ACT, Australia.