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

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Hilde Stawski (Creator)
Western Carolina University (WCU )
Web Site:
Mark Wilson

Abstract: Forensic DNA casework principally relies on the analysis of short tandem repeats (STRs) from nuclear DNA (nDNA). In cases where nDNA may not be suitable for analysis (i.e., highly degraded DNA or DNA present in quantities too low to obtain an STR profile), mitochondrial DNA (mtDNA) is an excellent alternative. MtDNA is a circular genome of approximately 16.5 kb, is maternally derived, and is present in thousands of copies per cell versus two copies of nuclear DNA. The combined higher copy number, circular shape of the genome and protection by the double membrane of the mitochondrion allows for a greater probability to recover sufficient mtDNA for typing of degraded samples. Presently, forensic analysts sequence two or three hypervariable (HV) regions found in the non-coding control region of the mtGenome, since sequencing of the entire mitochondrial genome (mtGenome) is rather costly and labor-intensive. Additionally, difficulties sequencing through homopolymeric regions, as well as the presence of lowlevel mixtures in samples, can add complexity to the analysis of mtDNA in casework when traditional Sanger sequencing methods are used. These issues can be addressed with Next Generation Sequencing (NGS) technologies. NGS enables deeper analysis of the genome for identification of low-level mixtures, since clonal populations of molecules originating from a single template strand are sequenced. Moreover, this technology allows for the more cost-effective sequencing of whole mtGenomes compared to Sanger methods, since more sequences are obtained for the same sample. By expanding mtDNA analysis to the entire mtGenome, a better resolution in distinguishing between haplotypes is established. 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 Illumina® Nextera® XT. This NGS library preparation kit is designed exclusively for use with Illumina® instrumentation and employs an engineered Transposome™ to randomly fragment and tag amplicons and small genomes with Illumina® specific adapters. After library preparation, samples were sequenced on the Illumina® MiSeq™. 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 Illumina® Nextera® XT and sequenced on the Illumina® MiSeq™. 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.

Additional Information

Language: English
Date: 2013
Forensic Science, Illumina MiSeq, Long PCR, Mitochondrial DNA, Next Generation Sequencing, Whole Genome Amplification
Mitochondrial DNA -- Analysis -- Data processing

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