Sequencing of mitochondrial genomes using the Precision ID mtDNA Whole Genome Panel

TY - JOUR

T1 - Sequencing of mitochondrial genomes using the Precision ID mtDNA Whole Genome Panel

AU - Pereira, Vania

AU - Longobardi, Antonio

AU - Børsting, Claus

PY - 2018/11

Y1 - 2018/11

N2 - Massively parallel sequencing offers a fast and cost-effective method for sequencing of the whole mtDNA genome. The Precision ID mtDNA Whole Genome Panel amplifies the entire mtDNA genome in two multiplex PCRs with 81 primer sets using the Ion AmpliSeq™ technology. In this study, the performance of the panel was evaluated by testing different amplification methods (two-in-one or conservative), the number of PCR cycles (21, 23, and 25), and different reaction volumes (recommended volume or half-volume). Furthermore, a dilution series, controlled mtDNA mixtures, and casework samples were also sequenced. The normalised read depths of the individual fragments were highly consistent irrespectively of the amplification methods or reaction volumes used. The sequencing output matched the mixture ratios of the controlled mtDNA mixtures indicating that the sequencing results were a loyal representation of the input DNA. Complete mtDNA profiles were generated from <10 pg genomic DNA. Seven fragments with relatively low read depths and large variations in read depth were identified. One of these fragments covered part of the control region and the hypervariable region II.

AB - Massively parallel sequencing offers a fast and cost-effective method for sequencing of the whole mtDNA genome. The Precision ID mtDNA Whole Genome Panel amplifies the entire mtDNA genome in two multiplex PCRs with 81 primer sets using the Ion AmpliSeq™ technology. In this study, the performance of the panel was evaluated by testing different amplification methods (two-in-one or conservative), the number of PCR cycles (21, 23, and 25), and different reaction volumes (recommended volume or half-volume). Furthermore, a dilution series, controlled mtDNA mixtures, and casework samples were also sequenced. The normalised read depths of the individual fragments were highly consistent irrespectively of the amplification methods or reaction volumes used. The sequencing output matched the mixture ratios of the controlled mtDNA mixtures indicating that the sequencing results were a loyal representation of the input DNA. Complete mtDNA profiles were generated from <10 pg genomic DNA. Seven fragments with relatively low read depths and large variations in read depth were identified. One of these fragments covered part of the control region and the hypervariable region II.

KW - Forensic genetics

KW - Massively parallel sequencing

KW - Mitochondrial DNA

KW - Next generation sequencing

KW - Precision ID mtDNA Whole Genome Panel

U2 - 10.1002/elps.201800088

DO - 10.1002/elps.201800088

M3 - Journal article

C2 - 30058717

SN - 0173-0835

VL - 39

SP - 2766

EP - 2775

JO - Electrophoresis

JF - Electrophoresis

IS - 21

ER -