title: Multiplex sequencing of pooled mitochondrial genomes-a crucial step toward biodiversity analysis using mito-metagenomics.
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.
authors.affiliation: China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China.
authors.forename: Min
authors.initials: M
authors.lastname: Tang
authors.affiliation: 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.
authors.forename: Meihua
authors.initials: M
authors.lastname: Tan
authors.affiliation: 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.
authors.forename: Guanliang
authors.initials: G
authors.lastname: Meng
authors.affiliation: China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China.
authors.forename: Shenzhou
authors.initials: S
authors.lastname: Yang
authors.affiliation: China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Su
authors.affiliation: China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China.
authors.forename: Shanlin
authors.initials: S
authors.lastname: Liu
authors.affiliation: China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Song
authors.affiliation: China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China.
authors.forename: Yiyuan
authors.initials: Y
authors.lastname: Li
authors.affiliation: China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Capital Normal University, Beijing 100094, China.
authors.forename: Aibing
authors.initials: A
authors.lastname: Zhang
authors.affiliation: China National GeneBank, BGI-Shenzhen, Beishan Road, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China xinzhou@genomics.cn.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhou
mesh_terms: D000818:Animals; D044822:Biodiversity; D003198:Computer Simulation; D004330:Drosophila / Q000235:genetics; D054629:Genome, Mitochondrial; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D056186:Metagenomics / Q000379:methods*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*