RNA-seq: technical variability and sampling.
RNA-seq: technical variability and sampling.
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DOI：
10.1186/1471-2164-12-293
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发表时间：
2011-06-06
期刊：
影响因子：
4.4
通讯作者：
Nuzhdin SV
中科院分区：
文献类型：
--
作者：
McIntyre LM;Lopiano KK;Morse AM;Amin V;Oberg AL;Young LJ;Nuzhdin SV
RNA-seq 正在彻底改变我们研究转录组的方式。无需事先了解基因转录本即可研究 mRNA。转录亚型的选择性剪接和先前未知的外显子的鉴定正在被报道。关于外显子使用差异、样本间剪接以及样本间定量差异的初步报告已开始浮出水面。据报道，生物变异比技术变异更大。此外，由于随机抽样，技术变化据报道符合预期。然而，处理技术变化的策略会根据程度的不同而有所不同。本手稿探讨了技术差异的大小以及抽样的作用。在这项研究中，分析了三个包含技术重复的独立 Solexa/Illumina 实验。当覆盖率较低时，技术重复之间的巨大分歧是显而易见的。当覆盖率低于每个核苷酸 5 个读数时，技术重复之间的外显子检测变化很大，并且当覆盖率较低时，基因表达的估计更有可能不一致。尽管在各个覆盖范围中都观察到表达估计存在很大分歧。技术变异性太高，不容忽视。技术差异导致低覆盖水平的外显子检测不一致。此外，即使覆盖水平很高，对转录本相对丰度的估计也可能大不相同。这可能是由于采样率较低，如果是这样，即使下一波技术产生更多的读数，它仍将作为实验设计中需要解决的问题持续存在。我们提供处理技术可变性的实用建议，而不会大幅增加成本。
RNA-seq is revolutionizing the way we study transcriptomes. mRNA can be surveyed without prior knowledge of gene transcripts. Alternative splicing of transcript isoforms and the identification of previously unknown exons are being reported. Initial reports of differences in exon usage, and splicing between samples as well as quantitative differences among samples are beginning to surface. Biological variation has been reported to be larger than technical variation. In addition, technical variation has been reported to be in line with expectations due to random sampling. However, strategies for dealing with technical variation will differ depending on the magnitude. The size of technical variance, and the role of sampling are examined in this manuscript. In this study three independent Solexa/Illumina experiments containing technical replicates are analyzed. When coverage is low, large disagreements between technical replicates are apparent. Exon detection between technical replicates is highly variable when the coverage is less than 5 reads per nucleotide and estimates of gene expression are more likely to disagree when coverage is low. Although large disagreements in the estimates of expression are observed at all levels of coverage. Technical variability is too high to ignore. Technical variability results in inconsistent detection of exons at low levels of coverage. Further, the estimate of the relative abundance of a transcript can substantially disagree, even when coverage levels are high. This may be due to the low sampling fraction and if so, it will persist as an issue needing to be addressed in experimental design even as the next wave of technology produces larger numbers of reads. We provide practical recommendations for dealing with the technical variability, without dramatic cost increases.
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