Massively parallel pyrosequencing of the mitochondrial genome with the 454 methodology in forensic genetics
Abstract
Results of sequencing of whole mitochondrial genome, HV1 and HV2 DNA with the second generation system (SGS) Roche 454 GS Junior were compared with results of Sanger sequencing and SNP typing with SNaPshot single base extension detected with MALDI-TOF and capillary electrophoresis. We investigated the performance of the software analysis of the data, reproducibility, ability to sequence homopolymeric regions, detection of mixtures and heteroplasmy as well as the implications of the depth of coverage. We found full reproducibility between samples sequenced twice with SGS. We found close to full concordance between the mtDNA sequences of 26 samples obtained with (1) the 454 SGS method using a depth of coverage above 100 and (2) Sanger sequencing and SNP typing. The discrepancies were primarily observed in homopolymeric regions. The 454 SGS method was able to sequence 95% of the reads correctly in homopolymers up to 4 bases, and up to 6 bases could be sequenced with similar success if the results were carefully, visually inspected. The 454 technology was able to detect mixtures or heteroplasmy of approximately 10%. We detected previously unreported heteroplasmy in the GM9947A component of the NIST human mitochondrial DNA SRM-2392 standard reference material.
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Different NGS platforms, such as Roche/454, Ion torrent and Illumina systems, have proven capable of sequencing the majority of STR loci in forensic science.
“Different NGS platforms, such as Roche/454, Ion torrent and Illumina systems, have proven capable of sequencing the majority of STR loci in forensic science (Fordyce et al., 2015; Mikkelsen et al., 2014; Van Neste et al., 2014).”
From Short tandem repeat profiling via next-generation sequencing for cell line authentication (2023) · cites this paper as Mikkelsen et al., 2014
STR-CE has a stutter ratio of 20%, which is often used for initial filtering.
“compared to a stutter ratio of 20% for STR-CE, which is often used for initial filtering (Brookes et al., 2012; Mikkelsen et al., 2014; Novroski et al., 2016).”
From Short tandem repeat profiling via next-generation sequencing for cell line authentication (2023) · cites this paper as Mikkelsen et al., 2014
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- Authors
- Mikkelsen, M., Frank-Hansen, R., Hansen, A. J., & Morling, N.
- Affiliations
- University of Copenhagen (DK)
- Corresponding author
- Mikkelsen, Martin
- Morling, Niels
- Venue
- Forensic Science International: Genetics
- Publisher
- Elsevier BV
- Published
- 2014-09
- Volume
- 12
- Pages
- 30-37
- ISSN
- 1872-4973 (ISSN-L)
- OpenAlex
- W2148342695
- Semantic Scholar
- 841ce7109ffd8efa915284f965ef5e96fa7cb86c
- Cited by
- 43
- References
- 30
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 1 · 2023: 3 · 2022: 1 · 2020: 3 · 2019: 3 · 2018: 6 · 2017: 8 · 2016: 7 · 2015: 2 · 2014: 7
- FWCI
- 5.24
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Forensic Science International Genetics (publishedVersion)
- PubMed (publishedVersion)
- Research at the University of Copenhagen (University of Copenhagen) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Forensic and Genetic Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic diversity and population structure
- Keywords
- mitochondrial DNA, pyrosequencing, forensic genetics, heteroplasmy, DNA mixtures, sequencing depth, reproducibility, Sanger sequencing comparison, mitochondrial DNA control region
- MeSH terms
- Base Sequence, DNA, Mitochondrial, Humans, Polymerase Chain Reaction, Sequence Analysis, DNA, DNA Primers, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Electrophoresis, Capillary, Polymorphism, Single Nucleotide, Forensic Genetics, Genome, Mitochondrial
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