Abstract
BackgroundMassively parallel sequencing (MPS) technologies have the capacity to sequence targeted regions or whole genomes of multiple nucleic acid samples with high coverage by sequencing millions of DNA fragments simultaneously. Compared with Sanger sequencing, MPS also can reduce labor and cost on a per nucleotide basis and indeed on a per sample basis. In this study, whole genomes of human mitochondria (mtGenome) were sequenced on the Personal Genome Machine (PGMTM) (Life Technologies, San Francisco, CA), the out data were assessed, and the results were compared with data previously generated on the MiSeqTM (Illumina, San Diego, CA). The objectives of this paper were to determine the feasibility, accuracy, and reliability of sequence data obtained from the PGM.Results24 samples were multiplexed (in groups of six) and sequenced on the at least 10 megabase throughput 314 chip. The depth of coverage pattern was similar among all 24 samples; however the coverage across the genome varied. For strand bias, the average ratio of coverage between the forward and reverse strands at each nucleotide position indicated that two-thirds of the positions of the genome had ratios that were greater than 0.5. A few sites had more extreme strand bias. Another observation was that 156 positions had a false deletion rate greater than 0.15 in one or more individuals. There were 31-98 (SNP) mtGenome variants observed per sample for the 24 samples analyzed. The total 1237 (SNP) variants were concordant between the results from the PGM and MiSeq. The quality scores for haplogroup assignment for all 24 samples ranged between 88.8%-100%.ConclusionsIn this study, mtDNA sequence data generated from the PGM were analyzed and the output evaluated. Depth of coverage variation and strand bias were identified but generally were infrequent and did not impact reliability of variant calls. Multiplexing of samples was demonstrated which can improve throughput and reduce cost per sample analyzed. Overall, the results of this study, based on orthogonal concordance testing and phylogenetic scrutiny, supported that whole mtGenome sequence data with high accuracy can be obtained using the PGM platform.
Highlights
Parallel sequencing (MPS) technologies have the capacity to sequence targeted regions or whole genomes of multiple nucleic acid samples with high coverage by sequencing millions of DNA fragments simultaneously
Short tandem repeat (STR) loci, single nucleotide polymorphisms (SNPs) and lineage markers (primarily residing within the mitochondrial DNA genome and Y chromosome) are the markers systems primarily used in forensic DNA typing and human identification [1,2,3,4,5,6,7]
Whole mtGenome sequencing was performed on the Personal Genome Machine (PGM) to determine its feasibility, accuracy, and reliability. These results were compared with sequence data previously generated on the MiSeq [19], and the findings demonstrated that reliable base calling can be obtained by the PGM system as well
Summary
Parallel sequencing (MPS) technologies have the capacity to sequence targeted regions or whole genomes of multiple nucleic acid samples with high coverage by sequencing millions of DNA fragments simultaneously. Compared with Sanger sequencing, MPS can reduce labor and cost on a per nucleotide basis and on a per sample basis. Short tandem repeat (STR) loci, single nucleotide polymorphisms (SNPs) and lineage markers (primarily residing within the mitochondrial DNA (mtDNA) genome and Y chromosome) are the markers systems primarily used in forensic DNA typing and human identification [1,2,3,4,5,6,7]. The mtDNA genome (mtGenome) is a marker of choice for human identification, especially where forensic biologic evidence contains too little or no nuclear DNA, such as a hair shaft without root, a fingernail and old bones. More discrimination power could be attained if more efficient and cost effective technologies allow expansion of genetic interrogation to the entire mtGenome
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
