Multiplexed DNA sequence capture of mitochondrial genomes using PCR products

Abstract

Background: To 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 findings: We 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 GA(II) instrument.

Conclusions/significance: This 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.

Figure 1. Overview of the capture-on-beads method.

On the left the production of the immobilized bait from two long range PCR products is shown; on the right the production of a pool of indexed libraries which are used in the capture (bottom). The eluted molecules can either be sequenced directly or first amplified and then sequenced. The bait is light red, mitochondrial DNA in the libraries is dark red, indices are shown in green and pink, adapters in gray. Thicker lines represent double stranded DNA while thinner lines represent single stranded DNA.

Figure 2. Number of reads sequenced (green bar) and aligned to the mitochondrial genome (red bar) for each sample.

Figure 3. Average (red squares) and minimum coverage (green squares) of the mitochondrial genome for each sample.

Figure 4. Coverage of each position across the whole mitochondrial genome, considering all the samples together.