Considering DNA damage when interpreting mtDNA heteroplasmy in deep sequencing data

Rathbun, Molly M.; McElhoe, Jennifer A.; Parson, Walther; Holland, Mitchell M.

doi:10.1016/j.fsigen.2016.09.008

Cited by 32 publications

(28 citation statements)

References 44 publications

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“…Cytosine deamination may be more readily identified than point heteroplasmy, because it causes specific base substitutions (C → T and G → A) at the ends of natural (non-amplified), degraded DNA fragments. Variants caused by cytosine deamination have been observed in forensic samples [65][66][67][68][69], and these random variants are not reproducible across PCR or library preparation events. Mixtures may be reproducible from the same DNA sample, yet they can be readily identified when the mixed samples have differing haplogroups, producing low frequency variants at haplogroup-diagnostic sites [44,63,[70][71][72][73][74][75].…”

Section: Numts In Forensic Applicationsmentioning

confidence: 99%

Interpreting NUMTs in forensic genetics: Seeing the forest for the trees

Marshall

Parson

2021

Forensic Science International: Genetics

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Nuclear mitochondrial DNA (mtDNA) segments (NUMTs) were discovered shortly after sequencing the first human mitochondrial genome. They have earlier been considered to represent archaic elements of ancient insertion events, but modern sequencing technologies and growing databases of mtDNA and NUMT sequences confirm that they are abundant and some of them phylogenetically young. Here, we build upon mtDNA/NUMT review articles published in the mid 2010 s and focus on the distinction of NUMTs and other artefacts that can be observed in aligned sequence reads, such as mixtures (contamination), point heteroplasmy, sequencing error and cytosine deamination. We show practical examples of the effect of the mtDNA enrichment method on the representation of NUMTs in the mapped sequence data and discuss methods to bioinformatically filter NUMTs from mtDNA reads.

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Section: Numts In Forensic Applicationsmentioning

confidence: 99%

Interpreting NUMTs in forensic genetics: Seeing the forest for the trees

Marshall

Parson

2021

Forensic Science International: Genetics

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“…DNA from sample 449, along with several others, were extracted from frozen blood samples up to 10 years old. DNA degradation is common in older stored samples, and often consists of cytosine bases undergoing deamination to uracil, which would be detected as a thymine in DNA sequencing that incorporates PCR enrichment or synthesis . It would be expected that the standard basecaller algorithms used with MinION data would recognize the uracil base as a thiamine as well, unless alternate basecalling rules were developed to specifically identify these base modifications.…”

Section: Resultsmentioning

confidence: 99%

Nanopore sequencing: An enrichment‐free alternative to mitochondrial DNA sequencing

2018

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Mitochondrial DNA sequence data are often utilized in disease studies, conservation genetics and forensic identification. The current approaches for sequencing the full mtGenome typically require several rounds of PCR enrichment during Sanger or MPS protocols followed by fairly tedious assembly and analysis. Here we describe an efficient approach to sequencing directly from genomic DNA samples without prior enrichment or extensive library preparation steps. A comparison is made between libraries sequenced directly from native DNA and the same samples sequenced from libraries generated with nine overlapping mtDNA amplicons on the Oxford Nanopore MinION™ device. The native and amplicon library preparation methods and alternative base calling strategies were assessed to establish error rates and identify trends of discordance between the two library preparation approaches. For the complete mtGenome, 16 569 nucleotides, an overall error rate of approximately 1.00% was observed. As expected with mtDNA, the majority of error was detected in homopolymeric regions. The use of a modified basecaller that corrects for ambiguous signal in homopolymeric stretches reduced the error rate for both library preparation methods to approximately 0.30%. Our study indicates that direct mtDNA sequencing from native DNA on the MinION™ device provides comparable results to those obtained from common mtDNA sequencing methods and is a reliable alternative to approaches using PCR‐enriched libraries.

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“…The combination of improved chemistry of the CRM kit and appropriate data processing with OREO allowed us to consider the whole control region to accurately call variants and heteroplasmies down to the level of 5%, identifying 197 different variants and twelve point heteroplasmies in this sample set. We note that other MPS-based approaches permit lower thresholds, thanks to different kit designs or deep sequencing [41,42]; however, for the general purposes of variant and heteroplasmy detection our limit of 5% seems sufficient.…”

Section: Discussionmentioning

confidence: 98%

Mitigating the effects of reference sequence bias in single-multiplex massively parallel sequencing of the mitochondrial DNA control region

Huszar

Wetton

Jobling

2019

Forensic Science International: Genetics

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Highlights mtDNA control region of 101 diverse samples amplified in a single reaction as 10 overlapping amplicons and sequenced via MPS. Primers create reference bias, compromising ability to call variants or heteroplasmy in primer-binding regions. Bioinformatic selection of overarching reads bypasses effects of proprietary primers and mitigates bias. Data processing permits accurate calling of variants, and heteroplasmies down to 5% level.

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Considering DNA damage when interpreting mtDNA heteroplasmy in deep sequencing data

Cited by 32 publications

References 44 publications

Interpreting NUMTs in forensic genetics: Seeing the forest for the trees

Interpreting NUMTs in forensic genetics: Seeing the forest for the trees

Nanopore sequencing: An enrichment‐free alternative to mitochondrial DNA sequencing

Mitigating the effects of reference sequence bias in single-multiplex massively parallel sequencing of the mitochondrial DNA control region

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