Unknown,Transcriptomics,Genomics,Proteomics

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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 M-bM-^@M-^XsPROFILERM-bM-^@M-^Y 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. mtDNA from 16 patient samples were analysed using Mitochip v2.0. Eight samples were DNA extractions directly from blood/bone marrow. The other eight samples were DNA extractions from primary cultured fibroblasts. Results were generated in GSEQ and reanalysed in sPROFILER for comparison.

ORGANISM(S): Homo sapiens

SUBMITTER: Gareth Price

PROVIDER: E-GEOD-29550 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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

Zamzami Mazin A MA Price Gareth R GR Taylor Robert W RW Blakely Emma L EL Oancea Iulia I Bowling Francis F Duley John A JA

BMC research notes 20111020

<h4>Background</h4>Developments in DNA resequencing microarrays include mitochondrial DNA (mtDNA) sequencing and mutation detection. Failure by the microarray to identify a base, compared to the reference sequence, is designated an 'N-call.' This study re-examined the N-call distribution of mtDNA samples sequenced by the Affymetrix MitoChip v.2.0, based on the hypothesis that N-calls may represent insertions or deletions (indels) in mtDNA.<h4>Findings</h4>We analysed 16 patient mtDNA samples usi ...[more]

PMID: 22011414

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

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

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