Zamzami Mazin A, Price Gareth R, Taylor Robert W, Blakely Emma L, Oancea Iulia, Bowling Francis, Duley John A
The University of Queensland, Brisbane, Australia.
BMC Res Notes. 2011 Oct 20;4:426. doi: 10.1186/1756-0500-4-426.
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.
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.
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.
DNA重测序微阵列技术的发展包括线粒体DNA（mtDNA）测序和突变检测。与参考序列相比，微阵列未能识别出的碱基被称为“N呼叫”。本研究基于“N呼叫可能代表mtDNA中的插入或缺失（indels）”这一假设，重新审视了由Affymetrix MitoChip v.2.0测序的mtDNA样本的N呼叫分布情况。
我们使用MitoChip分析了16例患者的mtDNA样本。当使用免费在线算法ResqMi或sPROFILER中的任何一种时，专有GSEQ软件的N呼叫显著减少。使用sPROFILER时，与GSEQ软件相比，N呼叫的减少对纯合或杂合突变水平没有影响，但ResqMi导致突变负荷发生显著变化，并产生更长的N细胞延伸。由于这些原因，未尝试使用ResqMi进行进一步分析。对sPROFILER较长的N呼叫延伸进行常规DNA测序，发现了7个插入和12个点突变。此外，对一个mtDNA样本的单碱基N呼叫分析发现了另外3个点突变。
我们的研究首次分析了MitoChipv2.0的GSEQ软件产生的N呼叫。通过将重点缩小到sPROFILER揭示的更长的N呼叫延伸，常规测序能够识别独特的插入和点突变。较短的N呼叫也含有点突变，但N呼叫中不存在缺失表明探针确认会影响结合，从而影响N呼叫。本研究支持了“GSEQ与sPROFILER结合使用时更能确定碱基”这一观点。
