Abstract

We present an improved de novo pipeline by combining long-range polymerase chain reaction (LR-PCR) and capture hybridization for enriching mitochondrial DNA to high-throughput sequencing. We test a new set of primers and hybridizing long-range library (LR-HY) with 112 mitochondrial genomes (MtG) representing three orders, 12 families, 54 genera, and 106 species of Amphibia. The primers are used for obtaining wide taxonomic MtG amplicons to sequence directly and/or make probes for closely related species. LR-HY is compared to standard hybridization. The primers successfully amplify 82 MtGs from all three order, all families, 92.6% (50/54) of the genera, and 74.5% (79/106) of the species, despite some DNA degradation and gene rearrangement. We observe a significantly negative correlation between sequence depth and gene variation. The pattern of highly variable regions is separately distributed in different regions within the length of < 4 kb in the 33-pooled sample. We demonstrate that using 2 kb libraries generate deeper sequence coverage in the highly variable loci than using 400 bp libraries. In total, the pipeline successfully recovers 83 complete and 14 almost complete MtGs from 53 of 54 genera, including 14 MtGs had rearranged protein-coding genes. This universal primers combined with LR-HY is an efficient way to enrich complete MtGs across the entire Amphibia.