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Open Access Highly Accessed Research article

Integrated shotgun sequencing and bioinformatics pipeline allows ultra-fast mitogenome recovery and confirms substantial gene rearrangements in Australian freshwater crayfishes

Han Ming Gan1*, Mark B Schultz2 and Christopher M Austin1

Author Affiliations

1 School of Science, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, 46150 Petaling Jaya, Selangor, Malaysia

2 Faculty of Medical and Dental Health Sciences, The University of Melbourne, Bio21 Research Institute, 30 Flemington Rd, Parkville, Victoria 3010, Australia

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BMC Evolutionary Biology 2014, 14:19  doi:10.1186/1471-2148-14-19

Published: 3 February 2014

Abstract

Background

Although it is possible to recover the complete mitogenome directly from shotgun sequencing data, currently reported methods and pipelines are still relatively time consuming and costly. Using a sample of the Australian freshwater crayfish Engaeus lengana, we demonstrate that it is possible to achieve three-day turnaround time (four hours hands-on time) from tissue sample to NCBI-ready submission file through the integration of MiSeq sequencing platform, Nextera sample preparation protocol, MITObim assembly algorithm and MITOS annotation pipeline.

Results

The complete mitochondrial genome of the parastacid freshwater crayfish, Engaeus lengana, was recovered by modest shotgun sequencing (1.2 giga bases) using the Illumina MiSeq benchtop sequencing platform. Genome assembly using the MITObim mitogenome assembler recovered the mitochondrial genome as a single contig with a 97-fold mean coverage (min. = 17; max. = 138). The mitogenome consists of 15,934 base pairs and contains the typical 37 mitochondrial genes and a non-coding AT-rich region. The genome arrangement is similar to the only other published parastacid mitogenome from the Australian genus Cherax.

Conclusions

We infer that the gene order arrangement found in Cherax destructor is common to Australian crayfish and may be a derived feature of the southern hemisphere family Parastacidae. Further, we report to our knowledge, the simplest and fastest protocol for the recovery and assembly of complete mitochondrial genomes using the MiSeq benchtop sequencer.

Keywords:
Freshwater crayfish; Mitochondrial genome characterization; Bench top sequencing; Engaeus; Parastacidae