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Nucleic Acids Res. 2014 Dec 16;42(22):e166. doi: 10.1093/nar/gku917. Epub 2014 Oct 7.

Multiplex sequencing of pooled mitochondrial genomes-a crucial step toward biodiversity analysis using mito-metagenomics.

Author information

1
China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China.
2
China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China University of Chinese Academy of Sciences, 19A Yuquan Road, Shijingshan District, Beijing 100094, China.
3
China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China China University of Geosciences, 388 Lumo Road, Wuhan 430074, China.
4
Capital Normal University, Beijing 100094, China.
5
China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China xinzhou@genomics.cn.

Abstract

The advent in high-throughput-sequencing (HTS) technologies has revolutionized conventional biodiversity research by enabling parallel capture of DNA sequences possessing species-level diagnosis. However, polymerase chain reaction (PCR)-based implementation is biased by the efficiency of primer binding across lineages of organisms. A PCR-free HTS approach will alleviate this artefact and significantly improve upon the multi-locus method utilizing full mitogenomes. Here we developed a novel multiplex sequencing and assembly pipeline allowing for simultaneous acquisition of full mitogenomes from pooled animals without DNA enrichment or amplification. By concatenating assemblies from three de novo assemblers, we obtained high-quality mitogenomes for all 49 pooled taxa, with 36 species >15 kb and the remaining >10 kb, including 20 complete mitogenomes and nearly all protein coding genes (99.6%). The assembly quality was carefully validated with Sanger sequences, reference genomes and conservativeness of protein coding genes across taxa. The new method was effective even for closely related taxa, e.g. three Drosophila spp., demonstrating its broad utility for biodiversity research and mito-phylogenomics. Finally, the in silico simulation showed that by recruiting multiple mito-loci, taxon detection was improved at a fixed sequencing depth. Combined, these results demonstrate the plausibility of a multi-locus mito-metagenomics approach as the next phase of the current single-locus metabarcoding method.

PMID:
25294837
PMCID:
PMC4267667
DOI:
10.1093/nar/gku917
[Indexed for MEDLINE]
Free PMC Article

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