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.
Highlights
Methods that enrich DNA samples for particular DNA sequences are important in order to allow efficient sequencing of targets that are present in complex mixtures of irrelevant DNA sequences
Methods that are able to ‘‘capture’’ relevant DNA sequences rely on hybridization of target sequences to probes that can be either in solution or immobilized on a surface (e.g. [3,4,5])
Bait production Two overlapping long-range PCR products encompassing the whole mitochondrial genome were produced as described [11]; DNA extracted from the saliva of one individual was used as the template
Summary
Methods that enrich DNA samples for particular DNA sequences are important in order to allow efficient sequencing of targets that are present in complex mixtures of irrelevant DNA sequences. We present a method where PCR products are used to capture targets for sequencing from pooled sequencing libraries of multiple individuals, using standard laboratory equipment. We apply this method to DNA pools of libraries from several human individuals from which we capture complete mitochondrial (mt) DNAs, a maternally inherited DNA molecule which is extensively studied in population genetics, medicine, forensics, and phylogenetics [8].
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