Sequencing the hypervariable regions of human mitochondrial DNA using massively parallel sequencing: Enhanced data acquisition for DNA samples encountered in forensic testing

Davis, Carey; Peters, Daniel R.; Warshauer, David H.; King, Jonathan L.; Budowle, Bruce

doi:10.1016/j.legalmed.2014.10.004

Cited by 26 publications

(9 citation statements)

References 9 publications

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“…In recent years, DNA sequencing technology has evolved from traditional Sanger sequencing of single reads to sequencing thousands of reads with high coverage in a massively parallel fashion with MPS technologies such as for analyzing human mtDNA [Sosa et al., , Parson et al. , ; Davis et al, ]. Contrary to the large‐fragment sequencing strategy employed mostly so far for MPS analysis of whole mt genomes [Sosa et al., ; Parson et al., ], here we introduce an MPS approach for analyzing the entire human mtDNA genome using 161 pairs of short amplicons (144–230 bp, average 200 bp) in two different primer pools.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, Davis et al. () used the MPS approach to analyze the hypervariable segments (HVS)‐I and II of human mtDNA using short amplicons in tissue and bone samples keeping with the previously discussed limitations of partial mtDNA analysis. However, the most established currently available MPS protocols for whole mtDNA genome sequencing are based on PCR amplification of large mtDNA fragments, typically several kilobases in size [Sosa et al., ; Parson et al., ], which fail when applied to degraded DNA as encountered in many mtDNA applications.…”

Section: Introductionmentioning

confidence: 98%

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Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine

et al. 2015

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Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long‐range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine

et al. 2015

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“…Since both the Illumina and Ion platforms are being marketed for mtDNA sequencing in forensic laboratories, it will be important to characterize the effect of the sequencing chemistry on the reporting of LHP in mtDNA profiles. In addition to alternative sequencing chemistries from which to evaluate LHP, NGS offers the ability to quantify heteroplasmic length variants with greater reliability [38,39], potentially increasing mtDNA haplotype resolution.…”

Section: Introductionmentioning

confidence: 99%

Impact of the sequencing method on the detection and interpretation of mitochondrial DNA length heteroplasmy

Sturk-Andreaggi

Parson

Allen

et al. 2020

Forensic Science International: Genetics

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“…Forensic mtDNA analysis typically uses PCR amplification and Sanger sequencing of the hypervariable regions of the control region, or D-loop (Kim et al, 2013;Lyons et al, 2013;Daud et al, 2014). However, massively parallel sequencing (MPS) offers the potential to obtain full mtDNA genomes to increase resolution between closely related haplogroups (Børsting et al, 2014;King et al, 2014;Yang et al, 2014;Davis et al, 2015;Parson et al, 2015;Zhou et al, 2016;Holland et al, 2017). Human identification cases that would most benefit from mtDNA analysis, such as long-term missing persons, human rights investigations and victims of disasters often involve DNA that is highly fragmented (<100 base pairs, bp).…”

Section: Introductionmentioning

confidence: 99%

Hybridization Enrichment to Improve Forensic Mitochondrial DNA Analysis of Highly Degraded Human Remains

2019

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Forensic mitochondrial DNA analysis of degraded human remains using PCR-based Sanger sequencing of the control region can be challenging when endogenous DNA is highly fragmented, damaged and at very low concentration. Hybridization enrichment coupled with massively parallel sequencing (MPS) offers an effective alternative for recovering DNA fragments as small as 30 base pairs (bp) from poorly preserved samples. Here, we apply this methodology on a range of degraded human skeletal remains that have previously been analyzed using PCR-based Sanger sequencing with variable success. Our results reaffirm the benefit of targeted enrichment for analysis of degraded remains and highlight the importance of using optimized library preparation and enrichment techniques. We provide an indication of the sequencing depth required to obtain full mtDNA genomes given the complexity of the library and confirm that a second enrichment and/or a very high sequencing effort may be required to obtain full mtDNA genomes for some degraded samples.

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Sequencing the hypervariable regions of human mitochondrial DNA using massively parallel sequencing: Enhanced data acquisition for DNA samples encountered in forensic testing

Cited by 26 publications

References 9 publications

Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine

Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine

Impact of the sequencing method on the detection and interpretation of mitochondrial DNA length heteroplasmy

Hybridization Enrichment to Improve Forensic Mitochondrial DNA Analysis of Highly Degraded Human Remains

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