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Analysis of the whole mitochondrial genome: translation of the Ion Torrent Personal Genome Machine system to the diagnostic bench?

ABSTRACT: Next-generation sequencing (NGS), an innovative sequencing technology that enables the successful analysis of numerous gene sequences in a massive parallel sequencing approach, has revolutionized the field of molecular biology. Although NGS was introduced in a rather recent past, the technology has already demonstrated its potential and effectiveness in many research projects, and is now on the verge of being introduced into the diagnostic setting of routine laboratories to delineate the molecular basis of genetic disease in undiagnosed patient samples. We tested a benchtop device on retrospective genomic DNA (gDNA) samples of controls and patients with a clinical suspicion of a mitochondrial DNA disorder. This Ion Torrent Personal Genome Machine platform is a high-throughput sequencer with a fast turnaround time and reasonable running costs. We challenged the chemistry and technology with the analysis and processing of a mutational spectrum composed of samples with single-nucleotide substitutions, indels (insertions and deletions) and large single or multiple deletions, occasionally in heteroplasmy. The output data were compared with previously obtained conventional dideoxy sequencing results and the mitochondrial revised Cambridge Reference Sequence (rCRS). We were able to identify the majority of all nucleotide alterations, but three false-negative results were also encountered in the data set. At the same time, the poor performance of the PGM instrument in regions associated with homopolymeric stretches generated many false-positive miscalls demanding additional manual curation of the data.

SUBMITTER: Seneca S

PROVIDER: S-EPMC4266735 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Analysis of the whole mitochondrial genome: translation of the Ion Torrent Personal Genome Machine system to the diagnostic bench?

Seneca Sara S Vancampenhout Kim K Van Coster Rudy R Smet Joél J Lissens Willy W Vanlander Arnaud A De Paepe Boel B Jonckheere An A Stouffs Katrien K De Meirleir Linda L

European journal of human genetics : EJHG 20140326 1

Next-generation sequencing (NGS), an innovative sequencing technology that enables the successful analysis of numerous gene sequences in a massive parallel sequencing approach, has revolutionized the field of molecular biology. Although NGS was introduced in a rather recent past, the technology has already demonstrated its potential and effectiveness in many research projects, and is now on the verge of being introduced into the diagnostic setting of routine laboratories to delineate the molecul ...[more]

PMID: 24667782

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Project description:BackgroundThe opioid system is involved in the control of pain, reward, addictive behaviors and vegetative effects. Opioids exert their pharmacological actions through the agonistic binding at opioid receptors and variation in the coding genes has been found to modulate opioid receptor expression or signaling. However, a limited selection of functional opioid receptor variants is perceived as insufficient in providing a genetic diagnosis of clinical phenotypes and therefore, unrestricted access to opioid receptor genetics is required.MethodsNext-generation sequencing (NGS) workflow was based on a custom AmpliSeq™ panel and designed for sequencing of human genes related to the opioid receptor group (OPRM1, OPRD1, OPRK1, SIGMA1, OPRL1) on an Ion PGM™ Sequencer. A cohort of 79 previously studied chronic pain patients was screened to evaluate and validate the detection of exomic sequences of the coding genes with 25 base pair exon padding. In-silico analysis was performed using SNP and Variation Suite® software.ResultsThe amplicons covered approximately 90% of the target sequence. A median of 2.54×106 reads per run was obtained generating a total of 35,447 nucleotide reads from each DNA sample. This identified approximately 100 chromosome loci where nucleotides deviated from the reference sequence GRCh37 hg19, including functional variants such as the OPRM1 rs1799971 SNP (118 A>G) as the most scientifically regarded variant or rs563649 SNP coding for μ-opioid receptor splice variants. Correspondence between NGS and Sanger derived nucleotide sequences was 100%.ConclusionResults suggested that the NGS approach based on AmpliSeq™ libraries and Ion PGM sequencing is a highly efficient mutation detection method. It is suitable for large-scale sequencing of opioid receptor genes. The method includes the variants studied so far for functional associations and adds a large amount of genetic information as a basis for complete analysis of human opioid receptor genetics and its functional consequences.

Project description: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.

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Analysis of the whole mitochondrial genome: translation of the Ion Torrent Personal Genome Machine system to the diagnostic bench?

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Analysis of the whole mitochondrial genome: translation of the Ion Torrent Personal Genome Machine system to the diagnostic bench?

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