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Insights into N-calls of mitochondrial DNA sequencing using MitoChip v2.0

ABSTRACT: Background: Developments in DNA resequencing microarrays include mitochondrial DNA (mtDNA) sequencing and mutation detection. During automated analysis to sequence mtDNA, bases can be identified as wildtype, variant, or there may be a failure to assign a base (N-call) when compared to the mtDNA reference sequence (rCRS). The purpose of our study was to re-examine the N-call distribution of mtDNA samples, based on the hypothesis that N-calls may represent insertions or deletions in mtDNA. Findings: We analysed mtDNA from 16 patient samples (8 blood/bone marrow origin and 8 from cultured fibroblasts) using the Affymetrix MitoChip v.2.0 which has 2 probe areas, one for sequencing compared to the rCRS and one for 500 common haplotypes. N-calls by the proprietary GSEQ software were significantly reduced when the freeware on-line algorithm ‘sPROFILER’ was utilized. Interestingly, this decrease in N-calls, in both sections of the MitoChip, had no effect on the homoplasmic or heteroplasmic mutation levels compared to GSEQ software. We then used conventional DNA sequencing to analyse continuous N-call stretches identified by sPROFILER, ranging from 2 to 22 bases, to identify changes resulting in base calling failures. Conclusions: Our study is the first to analyse N-calls produced from GSEQ software for the MitoChip v2.0. We found that by narrowing the focus to stretches of N-calls revealed by sPROFILER, conventional sequencing was able to identify unique deletions and insertions. This study also appeared to support the contention that the GSEQ is more capable of assigning bases when used in conjunction with sPROFILER.

ORGANISM(S): Homo sapiens

PROVIDER: GSE29550 | GEO | 2012/05/18

SECONDARY ACCESSION(S): PRJNA141313

REPOSITORIES: GEO

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Project description:Purpose: To develop a pipeline (Splice-Break) for high-resolution quantification of mtDNA deletions, provide a catalogue of human mtDNA deletion breakpoints, and evaluate mtDNA deletions in brains from subjects with psychiatric disorders. Methods: 93 samples from human postmortem brain and blood were obtained from the Southwest Brain Bank (SBB) and University of California, Irvine (UCI) Brain Bank. Total DNA was extracted from frozen homogenate tissue and mtDNA was amplified/enriched using a single long-range PCR. Mitochondrial amplicons were purified by bead purification to retain both wild-type and deleted molecules (i.e., no gel excision was performed). mtDNA-enriched PCR amplicons were prepared for sequencing using standard Illumina protocols for DNA. Samples were sequenced 150-mer paired-end reads, in multiplex (96x per lane), on an unpatterned flowcell (HiSeq 2500). Fastq files were processed using our Splice-Break pipeline for the detection and relative quantification of mtDNA deletions. Results: A catalogue of 4,489 putative mitochondrial DNA (mtDNA) deletions, including their frequency and relative read rate, was produced. Analyses of 93 samples from postmortem brain and blood found 1) the 4,977bp “common deletion” was neither the most frequent deletion nor the most abundant; 2) brain contained significantly more mtDNA deletions than blood; 3) many high frequency deletions were previously reported in MitoBreak, suggesting they are present at low levels in metabolically active tissues and are not exclusive to individuals with diagnosed mitochondrial pathologies; 4) many individual deletions (and cumulative deletion metrics) had significant and positive correlations with age; and 5) the highest deletion burdens were observed in a subset of subjects with major depressive disorder (MDD), and these subjects had mtDNA deletion levels at or above those detected in typical deletion pathologies (e.g., Kearns-Sayre syndrome (KSS) muscle). Conclusions: Collectively, these data suggest the Splice-Break pipeline can detect and quantify mtDNA deletions at a high level of resolution.

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Insights into N-calls of mitochondrial DNA sequencing using MitoChip v2.0

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