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

Han Gan,Christopher M Austin,Mark B Schultz

doi:10.1186/1471-2148-14-19

Han Gan, Christopher M Austin + Show 1 more

Open Access

https://doi.org/10.1186/1471-2148-14-19

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Abstract

BackgroundAlthough 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.ResultsThe 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.ConclusionsWe 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.

Highlights

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
A successful mitogenome assembly using the MiSeq benchtop sequencer has been demonstrated recently, the library preparation steps for sequencing on the MiSeq were not covered in sufficient details nor was MITObim implemented in the assembly pipeline [19]
Mitogenome assembly, coverage and composition A total of 4,761,100 paired-end reads amounting to approximately 1.2 giga bases of raw sequence data were generated from an E. lengana library

Summary

Introduction

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. The development of the MiSeq benchtop sequencer and the timely introduction of MITObim [14], a low computationally demanding software for the assembly of mitochondrial genomes using a novel baiting and iterative mapping approach, serves as an impetus for the growth in NGS-based mitogenome assemblies. Using a sample of the Australian freshwater crayfish Engaeus lengana [20], we contribute to the growing interest in mtDNA genome sequencing by providing a detailed protocol for the fastest recovery, assembly and annotation of mitogenome using the MiSeq personal genome sequencer, MITObim software and MITOS annotation web service

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