Multiplexed DNA Sequence Capture of Mitochondrial Genomes Using PCR Products

Abstract

BackgroundTo utilize the power of high-throughput sequencers, target enrichment methods have been developed. The majority of these require reagents and equipment that are only available from commercial vendors and are not suitable for the targets that are a few kilobases in length.Methodology/Principal FindingsWe describe a novel and economical method in which custom made long-range PCR products are used to capture complete human mitochondrial genomes from complex DNA mixtures. We use the method to capture 46 complete mitochondrial genomes in parallel and we sequence them on a single lane of an Illumina GAII instrument.Conclusions/SignificanceThis method is economical and simple and particularly suitable for targets that can be amplified by PCR and do not contain highly repetitive sequences such as mtDNA. It has applications in population genetics and forensics, as well as studies of ancient DNA.

Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus

“…The libraries were then quantified using a Bioanalyzer 2100 DNA 1000 assay (Agilent) and by quantitative PCR (qPCR) using the KAPA Library Quantification kit (Kapa Biosystems). The libraries were pooled in equimolar amounts across seven capture pools of 2 μg DNA each as previously described (Maricic et al 2010). The libraries created using Platinum Taq HiFi were split into two capture pools – the two dentine libraries formed the first pool and the six calculus libraries and the negative control formed the second pool.…”

“…DNA baits for the complete human mitochondrial genome were produced from modern DNA extracts of five individuals of diverse ancestry using previously described primers (Meyer et al 2007) and following previously reported protocols (Maricic et al 2010). Prior to capture, the baits were quantified using a Bioanalyzer 2100 DNA 1000 assay (Agilent).…”

Tracking niche variation over millennial timescales in sympatric killer whale lineages

“…Blank extractions were included to every five samples to monitor for contamination. To generate mitogenome sequences from recent (less than 200 years old) and ancient (greater than 1000 years old) samples, we used multiplexed DNA target enrichment hybridization capture coupled to high-throughput sequencing, using a published protocol [40] with a minor change, using heat instead of NaOH to release the captured DNA from the beads. Genomic DNA extract was built into blunt-ended libraries following Meyer & Kircher [41].…”

Complete mtDNA genomes of Filipino ethnolinguistic groups: a melting pot of recent and ancient lineages in the Asia-Pacific region

“…17 DNA libraries were sequenced on an Illumina GA IIx machine (Illumina Inc., San Diego, CA, USA) with post processing using Illumina software followed by the Improved Base Identification System. 18 Sequencing reads were mapped to the revised Cambridge Reference Sequence of the human mitochondrial genome (GenBank: NC012920.1) 19 using the program MIA, 20 implemented in an MPI-EVA sequence assembly-analyses pipeline for detecting mtDNA heteroplasmy 21 and low-level mutations 22 (Table 3) also does not support a discrete FEN vs FEnN grouping.…”