The time is now for ubiquitous forensic<scp>mtMPS</scp>analysis

Canale, Lauren C.; Parson, Walther; Holland, Mitchell M.

doi:10.1002/wfs2.1431

Cited by 7 publications

(8 citation statements)

References 116 publications

(257 reference statements)

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“…A gross assessment of MPS data quality was linked to the number and types of minor sequence variants reported by the analysis software (GM HTS), the generation of complete haplotypes, and the coverage of reads across the mitogenome. Minor variants consist of mixed sites due to heteroplasmy, damage sites, possible contamination, NUMTs (nuclear inserts of mitochondrial DNA), or random error [ 17 ]. An elevated number of minor variants may indicate the presence of DNA damage.…”

Section: Resultsmentioning

confidence: 99%

“…Unfortunately, sequencing the mitogenome using STS is labor-intensive, requires large quantities of DNA, and is a low-throughput method. Contrary to this, with advancements in massively parallel sequencing (MPS), analysis of the mitogenome can be performed relatively quickly with higher throughput and on samples that are routinely encountered in forensic cases, including hair shafts [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

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Routine Mitogenome MPS Analysis from 1 and 5 mm of Rootless Human Hair

Canale

McElhoe

Dimick

et al. 2022

Genes

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While hair shafts are a common evidence type in forensic cases, they are often excluded from DNA analysis due to their limited DNA quantity and quality. Mitochondrial (mt) DNA sequencing is the method of choice when working with rootless hair shaft fragments due to the elevated copy number of mtDNA and the highly degraded nature of nuclear (n) DNA. Using massively parallel sequencing (MPS) of the mitochondrial (mito) genome, we studied the impact of hair age (time since collection) and physical characteristics (hair diameter, medullary structure, and length of hair tested) on mtDNA recovery and MPS data quality. Hair shaft cuttings of 1 and 5 mm from hairs less than five years to 46 years of age from 60 donors were characterized microscopically. Mitogenome sequences were generated using the Promega PowerSeqTM Whole Mito System prototype kit and the Illumina MiSeq instrument. Reportable mitogenome sequences were obtained from all hairs up to 27 years of age (37 donors), with at least 98% of the mitogenome reported for more than 94% of the 74 hair samples analyzed; the minimum reported sequence was 88%. Furthermore, data from the 1 and 5 mm replicates gave concordant haplotypes. As expected, mtDNA yield decreased, mtDNA degradation increased, and mitogenome MPS data quality declined as the age of the hair increased. Hair diameter and medullary structure had minimal impact on yield and data quality. Our findings support that MPS is a robust and reliable method for routinely generating mitogenome sequences from 1 and 5 mm hair shaft samples up to 27 years of age, which is of interest to the forensic community, biological anthropologists, and medical geneticists.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Routine Mitogenome MPS Analysis from 1 and 5 mm of Rootless Human Hair

Canale

McElhoe

Dimick

et al. 2022

Genes

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“…Even with the benefits of WGS, the presence of microbial or exogenous DNA content will complicate sample processing, either requiring a higher amount of sequencing output (also increasing the price of the analysis) or a laborious pre-sequencing process [20]. Thus, many public forensic laboratories have been moving toward bringing IGG in-house through targeted MPS approaches that are more amenable to such challenging sample types, have a simple analysis pipeline, and utilize benchtop sequencers that are already known to the forensic community [21][22][23]. Specifically, the ForenSeq Kintelligence assay (Qiagen/Verogen, Inc.) was developed to address affordability and challenging samples in forensic laboratories.…”

Section: Introductionmentioning

confidence: 99%

Effects and considerations of multiplexing ForenSeq Kintelligence libraries with a negative control

Bali,

Cuenca,

Wallin

2024

Electrophoresis

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The negative template control or negative amplification control has been an essential component of the forensic DNA analysis workflow that helps monitor contamination. As such, the inclusion of a negative control in forensic DNA analysis has been a requirement for all laboratories audited under the FBI's Quality Assurance Standards. As massively parallel sequencing (MPS) becomes more conventional in forensic laboratories, considerations for the inclusion of a negative control in every sequencing run can be evaluated. Although the inclusion of a negative control in library preparation and the first sequencing run has a practical function, there is less utility for its inclusion in all subsequent sequencing runs for that library preparation. Although this is universal to all MPS assays, it is most relevant for an assay that has a low sample multiplexing capacity, such as the ForenSeq Kintelligence Kit (Qiagen/Verogen, Inc.). The ForenSeq Kintelligence Kit is an investigative genetic genealogy (IGG) sequencing‐based assay that targets 10,230 forensically relevant single‐nucleotide polymorphisms. The manufacturer recommends multiplexing 3 libraries per sequencing run, which includes controls. The purpose of this study was to investigate the effect of the inclusion of a negative control in every Kintelligence sequencing run. We observed that the library generated from a negative amplification control will take 7%–14% of the run output. The loss of sequencing space taken by a negative control decreased the available output for DNA‐containing samples, leading in some cases to allele or locus dropout and accompanying higher numbers of sixth to seventh order unknown associations in GEDmatch PRO.

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“…Readers should refer to Canale et al. (2021) for a review of mitogenome MPS analysis. Basic Protocol 3 provides a method for PCR amplification conditions and parameters for the entire mitogenome in a single multiplex commercially available from Promega.…”

Section: Introductionmentioning

confidence: 99%

“…Basic Protocol 2 provides a reliable, custom quantification assay to assess the number of mtDNA copies per unit volume in the DNA extract. Readers should refer to Canale et al (2021) for a review of mitogenome MPS analysis. Basic Protocol 3 provides a method for PCR amplification conditions and parameters for the entire mitogenome in a single multiplex commercially available from Promega.…”

Section: Introductionmentioning

confidence: 99%

Massively Parallel Sequencing of the Mitogenome from Human Hair Shafts in Forensic Investigations

Korber,

Canale,

Holland

2023

Current Protocols

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This article highlights methods used to perform DNA extraction, mitochondrial DNA quantification, multiplex PCR amplification, amplicon‐based massively parallel sequencing, and data analysis of the mitochondrial genome (mitogenome) from human hair shafts. The focus is on applications to forensic casework, but this set of protocols can be used for any purpose involving small cuttings (as small as 1 to 5 mm) of human hair shafts up to 40 years from the time of collection. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Extraction of mitochondrial DNA from human hair shaftsBasic Protocol 2: Quantification of mitochondrial DNA (copies/μl)Basic Protocol 3: Multiplex amplification of the mitogenomeBasic Protocol 4: Library preparation and sequencing of mitogenome ampliconsBasic Protocol 5: Data analysis of mitogenome haplotypes

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The time is now for ubiquitous forensicmtMPSanalysis

Cited by 7 publications

References 116 publications

Routine Mitogenome MPS Analysis from 1 and 5 mm of Rootless Human Hair

Routine Mitogenome MPS Analysis from 1 and 5 mm of Rootless Human Hair

Effects and considerations of multiplexing ForenSeq Kintelligence libraries with a negative control

Massively Parallel Sequencing of the Mitogenome from Human Hair Shafts in Forensic Investigations

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