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Open Access Technical Note

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

Mazin A Zamzami12, Gareth R Price3, Robert W Taylor4, Emma L Blakely4, Iulia Oancea15, Francis Bowling15 and John A Duley15*

Author Affiliations

1 The University of Queensland, Brisbane, Australia

2 King Abdulaziz University, Jeddah, Saudi Arabia

3 Pathology, Mater Health Services, Brisbane, Australia

4 Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, UK

5 Mater Medical Research Institute, Brisbane, Australia

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BMC Research Notes 2011, 4:426  doi:10.1186/1756-0500-4-426

Published: 20 October 2011

Abstract

Background

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.

Findings

We analysed 16 patient mtDNA samples using MitoChip. N-calls by the proprietary GSEQ software were significantly reduced when either of the freeware on-line algorithms ResqMi or sPROFILER was utilized. With sPROFILER, this decrease in N-calls had no effect on the homoplasmic or heteroplasmic mutation levels compared to GSEQ software, but ResqMi produced a significant change in mutation load, as well as producing longer N-cell stretches. For these reasons, further analysis using ResqMi was not attempted. Conventional DNA sequencing of the longer N-calls stretches from sPROFILER revealed 7 insertions and 12 point mutations. Moreover, analysis of single-base N-calls of one mtDNA sample found 3 other point mutations.

Conclusions

Our study is the first to analyse N-calls produced from GSEQ software for the MitoChipv2.0. By narrowing the focus to longer stretches of N-calls revealed by sPROFILER, conventional sequencing was able to identify unique insertions and point mutations. Shorter N-calls also harboured point mutations, but the absence of deletions among N-calls suggests that probe confirmation affects binding and thus N-calling. This study supports the contention that the GSEQ is more capable of assigning bases when used in conjunction with sPROFILER.