Highlights
- •Off-target haplotype (192 bp) was observed in MiSeq data of mtDNA in 16,209–16,400.
- •Two of four individuals and Control DNA 007 generated off-target haplotype.
- •Off-target haplotype matched to Poly_NumtS_430, HSA_NumtS_587, and others in BLAST.
- •Off-target haplotype was not contained in human reference sequence (GRCh38.p13).
- •Off-target haplotype was outside the modern human mtDNA in a phylogenetic tree.
Abstract
An increasing number of studies on massively parallel sequencing of mitochondrial
DNA (mtDNA) have been reporting identification of various types of noise or off-target
sequences. Herein, we report that an off-target haplotype (sequence length 192 bp)
observed in MiSeq data of mtDNA at nucleotide position 16,209–16,400 was likely caused
by polymorphic nuclear mitochondrial DNA sequences (NumtS). Buccal DNA samples from
Volunteers #001–004 and Control DNA 007 were amplified with our multiplex system of
the B (15,998−16,172), C (16,209−16,400), and E (30−289) regions using 2000 copies
of mtDNA. A sample index was added using a Nextera XT index kit, and MiSeq Reagent
Nano Kit v2 was used in 2 × 251 cycles on a MiSeq FGx. FASTQ files were analyzed by
CLC Genomics Workbench 21.0.3. The extracted SAM files were analyzed using our original
Excel macro to sum up the read counts as the phased variant calls for each region.
An off-target haplotype differing at 19 sites against the revised Cambridge reference
sequence was observed in Volunteer #001 (4 in 10 MiSeq data), Volunteer #002 (2 in
9), and Control DNA 007 (6 in 9). In a BLAST search, the sequence of the off-target
haplotype matched perfectly to three polymorphic NumtS (Poly_NumtS_430 [KM281528.1],
HSA_NumtS_587 [HE613849.1], and nuclear fossil [S80333.1]) and BAC clone of chromosome
11 (AC107937.2). The sequence also matched perfectly to a Filipino mtDNA (KC993973.1)
which was inferred as a chimeric sequence of mtDNA and the HSA_NumtS_587. The sequence
of the off-target haplotype was not contained in the latest human reference genome
sequence (GRCh38.p13). In a phylogenetic tree, the off-target haplotype was genetically
distant from modern human mtDNA and not directly connected to them. In conclusion,
observed off-target haplotype amplified by our multiplex system was likely caused
by Poly_NumtS_430 or HSA_NumtS_587.
Keywords
To read this article in full you will need to make a payment
Purchase one-time access:
Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online accessOne-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:
Subscribe to Forensic Science International: GeneticsAlready a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
References
- DNA commission of the international society for forensic genetics: revised and extended guidelines for mitochondrial DNA typing.Forensic Sci. Int.: Genet. 2014; 13: 134-142
- Developmental validation for sanger sequencing of HV1 and HV2 in mitochondrial DNA.Forensic Sci. Int.: Rep.2. 2020
- Second generation sequencing allows for mtDNA mixture deconvolution and high resolution detection of heteroplasmy.Croat. Med. J. 2011; 52: 299-313
- Reprint of: evaluation of next generation mtGenome sequencing using the ion torrent Personal Genome Machine (PGM).Forensic Sci. Int.: Genet. 2013; 7: 632-639
- Simultaneous detection of human mitochondrial DNA and nuclear-inserted mitochondrial-origin sequences (NumtS) using forensic mtDNA amplification strategies and pyrosequencing technology.J. Forensic Sci. 2014; 59: 1064-1073
- Development and assessment of an optimized next-generation DNA sequencing approach for the mtgenome using the Illumina MiSeq.Forensic Sci. Int.: Genet. 2014; 13: 20-29
- High-quality and high-throughput massively parallel sequencing of the human mitochondrial genome using the Illumina MiSeq.Forensic Sci. Int.: Genet. 2014; 12: 128-135
- Massively parallel pyrosequencing of the mitochondrial genome with the 454 methodology in forensic genetics.Forensic Sci. Int.: Genet. 2014; 12: 30-37
- Sequencing the hypervariable regions of human mitochondrial DNA using massively parallel sequencing: Enhanced data acquisition for DNA samples encountered in forensic testing.Leg. Med. (Tokyo). 2015; 17: 123-127
- Analysis of mixtures using next generation sequencing of mitochondrial DNA hypervariable regions.Croat. Med. J. 2015; 56: 208-217
- Massively parallel sequencing of complete mitochondrial genomes from hair shaft samples.Forensic Sci. Int.: Genet. 2015; 15: 8-15
- MPS analysis of the mtDNA hypervariable regions on the MiSeq with improved enrichment.Int. J. Leg. Med. 2017; 131: 919-931
- Investigation of length heteroplasmy in mitochondrial DNA control region by massively parallel sequencing.Forensic Sci. Int.: Genet. 2017; 30: 127-133
- Massively parallel sequencing-enabled mixture analysis of mitochondrial DNA samples.Int. J. Leg. Med. 2018; 132: 1263-1272
- Developmental validation of a Nextera XT mitogenome Illumina MiSeq sequencing method for high-quality samples.Forensic Sci. Int.: Genet. 2018; 34: 25-36
- Evaluation of the precision ID whole MtDNA genome panel for forensic analyses.Forensic Sci. Int.: Genet. 2018; 35: 21-25
- Evaluation of mitogenome sequence concordance, heteroplasmy detection, and haplogrouping in a worldwide lineage study using the Precision ID mtDNA Whole Genome Panel.Forensic Sci. Int.: Genet. 2019; 42: 244-251
- Validation of NGS for mitochondrial DNA casework at the FBI Laboratory.Forensic Sci. Int.: Genet. 2020; 44102151
- Impact of the sequencing method on the detection and interpretation of mitochondrial DNA length heteroplasmy.Forensic Sci. Int.: Genet. 2020; 44102205
- Estimation of the number of contributors to mixed samples of DNA by mitochondrial DNA analyses using massively parallel sequencing.Int. J. Leg. Med. 2020; 134: 101-109
- Characterization of background noise in MiSeq MPS data when sequencing human mitochondrial DNA from various sample sources and library preparation methods.Mitochondrion. 2020; 52: 40-55
- Bioinformatic removal of NUMT-associated variants in mitotiling next-generation sequencing data from whole blood samples.Electrophoresis. 2018; 39: 2785-2797
- A novel phylogenetic approach for de novo discovery of putative nuclear mitochondrial (pNumt) haplotypes.Forensic Sci. Int.: Genet. 2019; 43102146
- Distinguishing mitochondrial DNA and NUMT sequences amplified with the precision ID mtDNA whole genome panel.Mitochondrion. 2020; 55: 122-133
- Performance evaluation of a mitogenome capture and Illumina sequencing protocol using non-probative, case-type skeletal samples: implications for the use of a positive control in a next-generation sequencing procedure.Forensic Sci. Int.: Genet. 2017; 31: 198-206
- Polymorphic NumtS trace human population relationships.Hum. Genet. 2012; 131: 757-771
- The genomic landscape of polymorphic human nuclear mitochondrial insertions.Nucleic Acids Res. 2014; 42: 12640-12649
- Chapter 6 - Human nuclear mitochondrial sequences (NumtS).in: Gasparre G. Porcelli A.M. The Human Mitochondrial Genome. Academic Press, London2020: 131-143
Genome Reference Consortium, Frequently asked questions: how many individuals were sequenced for the human reference genome assembly? 〈https://www.ncbi.nlm.nih.gov/grc/help/faq/#human-reference-genome-individuals/〉, 2021 (Accessed Aug 10, 2021).
- A nuclear 'fossil' of the mitochondrial D-loop and the origin of modern humans.Nature. 1995; 378: 489-492
- Novel mitochondrial DNA insertion polymorphism and its usefulness for human population studies.Hum. Biol. 1996; 68: 847-854
- Numt identification and removal with RtN!.Bioinformatics. 2020; 36: 5115-5116
Applied Biosystems, Solutions for mtDNA analysis. 〈https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FGSD%2FFlyers%2Fprecisionid-mtDNA-panel-flyer.pdf〉, 2019 (Accessed Nov 16, 2021).
Verogen, Universal Analysis Software v2.0 reference guide (Rev. E). 〈https://verogen.com/wp-content/uploads/2021/05/universal-analysis-software-v2–0-reference-guide-vd2019002-e.pdf/〉, 2021 (Accessed Nov 16, 2021).
- Reanalysis and revision of the cambridge reference sequence for human mitochondrial DNA.Nat. Genet. 1999; 23: 147
Illumina, 16S metagenomic sequencing library preparation (15044223 Rev. B). 〈https://jp.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf/〉, 2013 (Accessed Mar 4, 2022).
QIAgen, Manual for CLC Genomics Workbench 21.0.3 Windows, macOS and Linux. 〈https://digitalinsights.qiagen.com/technical-support/manuals/〉, 2021 (Accessed Feb 22, 2021).
- AQME: a forensic mitochondrial DNA analysis tool for next-generation sequencing data.Forensic Sci. Int.: Genet. 2017; 31: 189-197
- BioEit: a user friendly biological sequence aligment editor and analysis program for windows 95/98/NT.Nucleic Acids Symp. Ser. 1999; 41: 95-98
- MEGA X: molecular evolutionary genetics analysis across computing platforms.Mol. Biol. Evol. 2018; 35: 1547-1549
- The neighbor-joining method: a new method for reconstructing phylogenetic trees.Mol. Biol. Evol. 1987; 4: 406-425
- Complete mtDNA genomes of Filipino ethnolinguistic groups: a melting pot of recent and ancient lineages in the Asia-Pacific region.Eur. J. Hum. Genet. 2014; 22: 228-237
- Numt-mediated double-strand break repair mitigates deletions during primate genome evolution.PLoS Genet. 2008; 4e1000237
- HmtDB 2016: data update, a better performing query system and human mitochondrial DNA haplogroup predictor.Nucleic Acids Res. 2017; 45: D698-D706
- Multiplexed DNA sequence capture of mitochondrial genomes using PCR products.PLoS One. 2010; 5e14004
- A mitochondrial genome sequence of a hominin from Sima de los Huesos.Nature. 2014; 505: 403-406
- Human origins in a southern African palaeo-wetland and first migrations.Nature. 2019; 575: 185-189
- The complete mitochondrial DNA genome of an unknown hominin from southern Siberia.Nature. 2010; 464: 894-897
- Tree of life reveals clock-like speciation and diversification.Mol. Biol. Evol. 2015; 32: 835-845
- Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells.J. Cell. Physiol. 1988; 136: 507-513
- Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell.Exp. Cell Res. 1991; 196: 137-140
- Quantification of mitochondrial DNA copy number in suspected cancer patients by a well optimized ddPCR method.Biomol. Detect. Quantif. 2017; 13: 32-39
Article info
Publication history
Published online: May 04, 2022
Accepted:
May 1,
2022
Received in revised form:
March 12,
2022
Received:
September 17,
2021
Identification
Copyright
© 2022 Elsevier B.V. All rights reserved.
