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Understanding the genomes of endangered animal species

Understanding the genomes of endangered animal species

The wild population of the red-spotted grouper Epinephelus akaara (E. akaara), an economically important marine fish species, has significantly declined in recent decades and is now a threatened species. Genomic information would aid in understanding E. akaara diversity and population dynamics, and thereby support its conservation and management; however, a reference genome has yet to be generated1.

Ge and colleagues combined nanopore whole-genome sequencing with Hi-C to produce a reference genome for E. akaara1. Using the Oxford Nanopore GridION sequencing platform, 106.29 Gb of filtered data was obtained, assembled with Canu, and polished with nanopolish and Pilon, to achieve a final genome assembly of 1.135 Gb in 2,055 contigs, which was 96.8% complete. Repetitive sequences comprised 43.02% of the genome, of which transposons accounted for 16.73%. In total, 23,923 protein-coding genes were identified, of which up to 99.5% were functionally annotated.

Chondrichthyes (sharks, rays, skates, and chimaeras) are vital for the maintenance of oceanic ecosystems, yet, of the ~1,178 chondrichthyan species known, 25% are threatened with extinction due to global overfishing and exploitation resulting from the demand for shark-and ray-derived products2. A lack of ecological data for these species has hampered their effective conservation3. Current molecular ecological studies for taxonomic identification and population analysis of chondrichthyans are based on PCR amplification and sequencing of DNA “barcodes” from the mitochondrial and nuclear genomes (e.g. cytochrome oxidase I, 16S rRNA, and Recombination-Activating Gene I). This process requires a priori sequence information, technical expertise, time, and a laboratory infrastructure.

‘Our genome assembly results demonstrate that long sequence reads produced by nanopore sequencing can be effectively used for genome assembly’1

Demonstrating a method for accurate taxonomic identification, and for addressing the paucity of chondrichthyan genomic information, Shaili Johri and colleagues performed direct sequencing of genomic DNA on the portable MinION platform — reducing processing time, avoiding amplification bias, negating the need for a priori sequence knowledge, and allowing local analysis on a portable computer3. The team performed “genome skimming” — deep sequencing of high copy number fractions of the genome — of a shark fin specimen. With 74,536 reads, they employed two analysis workflows for taxonomic identification (Figure 1). The first workflow, recommended for urgent in-field analysis, involved mapping reads to published chondrichthyan mitogenomes, which enabled assembly of a complete mitochondrial contig of 16,675 bp with mean 19.4x depth of coverage. The second workflow involved assembly and subsequent alignment to mitochondrial and nuclear sequences for contig annotation; this provided an almost complete mitogenome as well as 1–50x depth of coverage of nuclear regions. Phylogenetic analysis based on the recovered genomic regions identified the unknown sample as Carcharhinus falciformis, a CITES Appendix II listed protected species, for which trade must be controlled.

‘These data were acquired in a very brief time frame, without any amplification bias, and with minimal infrastructure compared to PCR based studies’3

Figure 6: The two workflows used for sequence analysis by Johri et al; one recommended for in-field or forensic applications with no connectivity and computing power of an average laptop, and the second for detailed laboratory analyses, with connectivity and high computing power. Taken from Johri et al. (2019)48 and available under Creative Commons license (creativecommons.org/ licenses/by/4.0).

References

  1. Ge, H. et al. (2019). De novo assembly of a chromosome-level reference genome of red-spotted grouper (Epinephelus akaara) using nanopore sequencing and Hi-C. Mol Ecol Resour doi: 10.1111/1755-0998.13064.
  2. Johri, S. Personal communication with Oxford Nanopore Technologies on 06 September 2019.
  3. Johri, S. et al. (2019). ‘Genome skimming’ with the MinION hand-held sequencer identifies CITES-listed shark species in India’s exports market. Sci Rep 9(1):4476.
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