Preparing whole genome human mitochondrial DNA libraries for next generation sequencing using Illumina Nextera XT

In forensic casework, amplification of challenging samples such as hair and aged bone is often performed differently than that of reference samples (buccal swabs, blood, etc.) due to the higher possibility of DNA degradation and limited mtDNA concentrations. For this study, two sample preparation approaches were developed including one method for robust reference samples, and one method for forensically relevant challenging samples.;For NGS analysis of reference samples, DNA was extracted from buccal swabs obtained from eight donors. A long PCR approach, which refers to the amplification of DNA fragments of a size that may not be amplified using conventional PCR reagents, was successfully performed on these DNA extracts using a highly processive polymerase mixture and novel primer pairs to amplify the mtGenome in two independent PCR reactions, with overlap at the noncoding region. These samples were subsequently processed with IlluminaRTM NexteraRTM XT. This NGS library preparation kit is designed exclusively for use with IlluminaRTM instrumentation and employs an engineered Transposome(TM) to randomly fragment and tag amplicons and small genomes with IlluminaRTM specific adapters. After library preparation, samples were sequenced on the IlluminaRTM MiSeq(TM). This method generated whole mtGenome NGS data, which accurately reflected the Sanger sequence.;For analysis of challenging samples, DNA was extracted from 2 cm fragments of hair shafts from a subset of the same donors, using an optimized DNA extraction protocol. Whole genome amplification (WGA) was performed on these extracts with four different commercially available WGA kits. WGA allows for pre-amplification of the entire mtGenome without the need for any additional primer design, after which the resulting DNA can be used for downstream applications. This potentially provides the forensic analyst with an increase in DNA template, resulting in a higher possibility of obtaining useful data from a casework sample. The increase in mtDNA copy number was assessed with a human mtDNA specific qPCR assay. A subset of the samples before and after WGA was amplified using a targeted multiplex PCR approach. This product, in addition to a subset of WGA product that was not PCR amplified after WGA, was prepared with IlluminaRTM NexteraRTM XT and sequenced on the IlluminaRTM MiSeq(TM).;This research effort generated a protocol for obtaining whole mtGenome NGS data from reference samples such as buccal swabs. In addition, preliminary data was generated for future studies designed to obtain whole mtGenome NGS data from challenging sample types. (Abstract shortened by UMI.).