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

Huszar, Tunde I.; Wetton, Jon H.; Jobling, Mark A.

doi:10.1016/j.fsigen.2019.01.008

Cited by 9 publications

(6 citation statements)

References 42 publications

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“…The amplicon settings, which define the data ranges for each of the assay amplicons, were initially set at 16013-16126, 16116-16225, 16224-16408, 16387-16486, 16474-30, 16555-152, 136-257, 246-364, 342-436, and 429-592, based on consultation with the vendor. These ranges sit just inside the primer regions for each of the 10 amplicons of the PowerSeq mtDNA CR Nested System, as analyzing data within the PCR primer binding regions can produce false point heteroplasmies [30]. Upon examination of the mixture data, the amplicon 3 settings were updated to 16223-16408 (for the reasons described in Section 3.3, below).…”

Section: Discussionmentioning

confidence: 99%

Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

Brandhagen

Just

Irwin

2020

Forensic Science International: Genetics

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As a first step towards integrating next generation sequencing (NGS) technology into the FBI Laboratory's operational casework, the PowerSeq™ CRM Nested System, an NGS-based mitochondrial DNA (mtDNA) control region assay, was developmentally and internally validated. The validation studies were conducted in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) Validation Guidelines for Forensic DNA Analysis Methods, and the FBI's Quality Assurance Standards (QAS) for Forensic DNA Testing Laboratories. The assay was shown to be highly reproducible, with variant frequencies across intra and inter-run replicates of the same sample differing, on average, by just 0.3% for substitutions and point heteroplasmies and 1.5% for insertions and deletions. The assay was also shown to be extremely sensitive, yielding complete control region sequence data from as few as 2000 copies of mtDNA. This is a more than 20-fold increase in sensitivity when compared to the FBI Laboratory's current Sanger sequencing-based protocols and, based on mtDNA quantitation values of samples routinely encountered in mtDNA casework, suggests that the percentage of questioned samples from which full control region data can be recovered will increase from our current 20% to approximately 90% success with NGS technology. In addition, the assay requires on average only 30% of the extract volume typically required to develop control region profiles from degraded samples via Sanger sequencing. Overall, these studies establish the reliability of the PowerSeq™ CRM Nested System for accurate mtDNA control region typing and can serve as a model for laboratories seeking to validate NGS protocols for forensic mtDNA analysis.

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Section: Discussionmentioning

confidence: 99%

Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

Brandhagen

Just

Irwin

2020

Forensic Science International: Genetics

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“…The remaining positions were usually of low quality and were counted as false negatives, because they were missed in at least one sequence sample (see below). We reasoned that a reference allele bias effect could impact the quality of variant calling, especially in the highly polymorphic and repetitive control region [35]. Therefore, we performed the same variant calling procedure using two alternative references: a Thoroughbred breed more closely related to H25 and H27, and the Twilight horse (a Thoroughbred) used for the equine reference genome.…”

Section: Mitochondrial Dna Variants Callingmentioning

confidence: 99%

A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

2020

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Background Mitochondrial DNA is remarkably polymorphic. This is why animal geneticists survey mitochondrial genomes variations for fundamental and applied purposes. We present here an approach to sequence whole mitochondrial genomes using nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA using an exonuclease treatment and on the amplification of circular mitochondrial DNA using a multiple displacement amplification step. Results We optimized each preparative step to obtain a 100 million-fold enrichment of horse mitochondrial DNA relative to nuclear DNA. We sequenced these amplified mitochondrial DNA using nanopore sequencing technology and obtained mitochondrial DNA reads that represented up to half of the sequencing output. The sequence reads were 2.3 kb of mean length and provided an even coverage of the mitochondrial genome. Long-reads spanning half or more of the whole mtDNA provided a coverage that varied between 118X and 488X. We evaluated SNPs identified using these long-reads by Sanger sequencing as ground truth and found a precision of 100.0%; a recall of 93.1% and a F1-score of 0.964 using the Twilight horse mtDNA reference. The choice of the mtDNA reference impacted variant calling efficiency with F1-scores varying between 0.947 and 0.964. Conclusions Our method to amplify mtDNA and to sequence it using the nanopore technology is usable for mitochondrial DNA variant analysis. With minor modifications, this approach could easily be applied to other large circular DNA molecules.

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“…Some approaches to MSA primer design, like the JCVI primer designer, often make use of reference sequences ( Li et al 2008 , 2012 ). One problem with this approach is reference sequences introduce a primer matching bias within interspecies comparisons favoring sequences with the most matches to the given reference ( Huszar et al 2019 ). Within the human microbiome, where intraspecies genetic diversity and mutation rates may be higher, the reference sequence may not remain a valid basis of comparison for any particular experiment ( Ramiro et al 2020 ).…”

Section: Resultsmentioning

confidence: 99%

DeGenPrime provides robust primer design and optimization unlocking the biosphere

Fulghum,

Tanker,

White

2024

Bioinformatics Advances

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Motivation Polymerase chain reaction (PCR) is the world’s most important molecular diagnostic with applications ranging from medicine to ecology. PCR can fail because of poor primer design. The nearest-neighbor thermodynamic properties, picking conserved regions, and filtration via penalty of oligonucleotides form the basis for good primer design. Results DeGenPrime is a console-based high quality PCR primer design tool that can utilize MSA formats and degenerate bases expanding target range for a single primer set. Our software utilizes thermodynamic properties, filtration metrics, penalty scoring, and conserved region finding of any proposed primer. It has degeneracy, repeated k-mers, relative GC content, and temperature range filters. Minimal penalty scoring is included according to secondary structure self-dimerization metrics, GC clamping, tri- and tetra-loop hairpins and internal repetition. We compared PrimerDesign-M, DegePrime, ConsensusPrimer, and DeGenPrime on acceptable primer yield. PrimerDesign-M, DegePrime, and ConsensusPrimer provided 0%, 11%, and 17% yield respectively for alternative iron nitrogenase (anfD) gene target. DeGenPrime successfully identified quality primers within the conserved regions of the T4-like phage major capsid protein (g23), conserved regions of molybdenum-based nitrogenase (nif), and its alternatives vanadium (vnf) and iron (anf) nitrogenase. DeGenPrime provides a universal and scalable primer design tool for the entire tree of life. Availability DeGenPrime is written in C ++ and distributed under a BSD-3-Clause license. The source code for DeGenPrime is freely avaliable on www.github.com/raw-lab/degenprime. Supplementary information Supplementary data are available at Bioinformatics Advances online.

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

Cited by 9 publications

References 42 publications

Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

DeGenPrime provides robust primer design and optimization unlocking the biosphere

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