Evaluation of GeneMarker® HTS for improved alignment of mtDNA MPS data, haplotype determination, and heteroplasmy assessment.

Existing software has not allowed for effective alignment of mitochondrial (mt) DNA sequence data generated using a massively parallel sequencing (MPS) approach, combined with the ability to perform a detailed assessment of the data. The regions of sequence that are typically difficult to align are homopolymeric stretches, isolated patterns of SNPs (single nucleotide polymorphisms), and INDELs (insertions/deletions). A custom software solution, GeneMarker® HTS, was developed and evaluated to address these limitations, and to provide a user-friendly interface for forensic practitioners and others interested in mtDNA analysis of MPS data. GeneMarker® HTS generates an exportable consensus mtDNA sequence that produces phylogenetically correct SNP and INDEL calls using a customizable motif-based alignment algorithm. Sequence data from 500 individuals, with various alignment asymmetries and levels of heteroplasmy, were used to assess the software. Accuracy in producing mtDNA haplotypes, the ability to correctly identify low-level heteroplasmic sequence variants, and the user-based features of the software were evaluated. Analyzed sequences yielded correct mtDNA haplotypes, and heteroplasmic variants were properly identified with minimal manual interpretation. The software offers numerous user-defined parameters for filtering the data that address the interests of researchers and practitioners, and provides multiple options for viewing and navigating through the data.

[1]  L. Bachmann,et al.  Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—a baiting and iterative mapping approach , 2013, Nucleic acids research.

[2]  E. Salisbury.  POLYMORPHISM IN THE FLOWER OF SILENE MARITIMA. , 1912 .

[3]  A. Klindworth,et al.  Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies , 2012, Nucleic acids research.

[4]  R. Kaneva,et al.  Validation of an NGS Approach for Diagnostic BRCA1/BRCA2 Mutation Testing , 2015, Molecular Diagnosis & Therapy.

[5]  Bruce Budowle,et al.  mitoSAVE: mitochondrial sequence analysis of variants in Excel. , 2014, Forensic science international. Genetics.

[6]  M. Holland,et al.  Second generation sequencing allows for mtDNA mixture deconvolution and high resolution detection of heteroplasmy , 2011, Croatian medical journal.

[7]  M. Holland,et al.  Mitochondrial DNA sequence analysis of human skeletal remains: identification of remains from the Vietnam War. , 1993, Journal of forensic sciences.

[8]  W R Mayr,et al.  DNA Commission of the International Society for Forensic Genetics: revised and extended guidelines for mitochondrial DNA typing. , 2014, Forensic science international. Genetics.

[9]  D. Turnbull,et al.  Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA , 1999, Nature Genetics.

[10]  W. Parson,et al.  Massively parallel sequencing of complete mitochondrial genomes from hair shaft samples. , 2015, Forensic science international. Genetics.

[11]  F. Sanger,et al.  Sequence and organization of the human mitochondrial genome , 1981, Nature.

[12]  P. Jaccard.  THE DISTRIBUTION OF THE FLORA IN THE ALPINE ZONE.1 , 1912 .

[13]  P. Ivanov,et al.  Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II , 1996, Nature Genetics.

[14]  G. Millat,et al.  Evaluation of a new NGS method based on a custom AmpliSeq library and Ion Torrent PGM sequencing for the fast detection of genetic variations in cardiomyopathies. , 2014, Clinica chimica acta; international journal of clinical chemistry.

[15]  W. Parson,et al.  Consistent treatment of length variants in the human mtDNA control region: a reappraisal , 2006, International Journal of Legal Medicine.

[16]  Rebecca S. Just,et al.  Mitochondrial DNA heteroplasmy in the emerging field of massively parallel sequencing , 2015, Forensic science international. Genetics.

[17]  J. Irwin,et al.  Concordance and reproducibility of a next generation mtGenome sequencing method for high-quality samples using the Illumina MiSeq. , 2016, Forensic science international. Genetics.

[18]  T. A. Sivakumaran,et al.  Performance Evaluation of the Next-Generation Sequencing Approach for Molecular Diagnosis of Hereditary Hearing Loss , 2013, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery.

[19]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[20]  Jiang Li,et al.  MitoSeek: extracting mitochondria information and performing high-throughput mitochondria sequencing analysis , 2013, Bioinform..

[21]  M S Waterman,et al.  Identification of common molecular subsequences. , 1981, Journal of molecular biology.

[22]  O. Drummer,et al.  The contribution of DNA to the disaster victim identification (DVI) effort. , 2011, Forensic science international.

[23]  Anita Brandstätter,et al.  Generating population data for the EMPOP database - an overview of the mtDNA sequencing and data evaluation processes considering 273 Austrian control region sequences as example. , 2007, Forensic science international.

[25]  Mitchell M Holland,et al.  Development and assessment of an optimized next-generation DNA sequencing approach for the mtgenome using the Illumina MiSeq. , 2014, Forensic science international. Genetics.

[26]  Ernesto Picardi,et al.  MToolBox: a highly automated pipeline for heteroplasmy annotation and prioritization analysis of human mitochondrial variants in high-throughput sequencing , 2014, Bioinform..

[27]  Steven A. Krieger.  Why Our Justice System Convicts Innocent People, and the Challenges Faced by Innocence Projects Trying to Exonerate Them , 2011 .

[28]  Anton Nekrutenko,et al.  Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA , 2014, Proceedings of the National Academy of Sciences.

[29]  Walther Parson,et al.  Improved visibility of character conflicts in quasi-median networks with the EMPOP NETWORK software , 2014, Croatian medical journal.