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)
- Institution
- Western Carolina University (WCU )
- Web Site: http://library.wcu.edu/
- Advisor
- 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.
Preparing whole genome human mitochondrial DNA libraries for next generation sequencing using Illumina Nextera XT
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Created on 12/1/2013
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2013
- Keywords
- Forensic Science, Illumina MiSeq, Long PCR, Mitochondrial DNA, Next Generation Sequencing, Whole Genome Amplification
- Subjects
- Mitochondrial DNA -- Analysis -- Data processing