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

Zamzami, Mazin A., Price, Gareth R., Taylor, Robert W., Blakely, Emma L., Oancea, Iulia, Bowling, Francis and Duley, John A. (2011) Insights into N-calls of mitochondrial DNA sequencing using MitoChip v2.0. BMC Research Notes, 4 426.1-426.8. doi:10.1186/1756-0500-4-426


Author Zamzami, Mazin A.
Price, Gareth R.
Taylor, Robert W.
Blakely, Emma L.
Oancea, Iulia
Bowling, Francis
Duley, John A.
Title Insights into N-calls of mitochondrial DNA sequencing using MitoChip v2.0
Journal name BMC Research Notes   Check publisher's open access policy
ISSN 1756-0500
Publication date 2011-10-20
Sub-type Article (original research)
DOI 10.1186/1756-0500-4-426
Open Access Status DOI
Volume 4
Start page 426.1
End page 426.8
Total pages 8
Place of publication London, United Kingdom
Publisher BioMed Central
Collection year 2012
Language eng
Formatted 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.
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Article number 426

Document type: Journal Article
Sub-type: Article (original research)
Collections: Mater Research Institute-UQ (MRI-UQ)
Official 2012 Collection
School of Medicine Publications
School of Pharmacy Publications
 
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Created: Tue, 17 Jan 2012, 11:10:08 EST by Dr John Duley on behalf of School of Pharmacy