Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data
Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data
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DOI：
10.1261/rna.046060.114
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发表时间：
2015-02-01
期刊：
影响因子：
4.5
通讯作者：
Lenburg, Marc E.
中科院分区：
文献类型：
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作者：
Campbell, Joshua D.;Liu, Gang;Lenburg, Marc E.
小RNA测序可用于获得microRNA转录组中前所未有的大量细节。测序碱基技术的相对高成本和低通量可以通过使用多路复用来潜在地抵消。然而，多路复用涉及增加测序样本数量和减少每个样本的读取数量（即较低的覆盖深度）之间的权衡。为了评估由于多路复用而导致的不同测序深度对microRNA差异表达和检测的影响，我们使用Illumina HiSeq 2000对临床环境中收集的肺组织样本的小RNA进行了测序，每通道分别进行1、3、6、9或12个样本的多路复用。正如预期的那样，每个样本获得的读取数量随着多路复用中样本数量的增加而减少。此外，在归一化后，不同复片中包含的重复样本高度相关（R = 0.97）。当检测各组样品之间的差异microRNA表达时，平均表达量为>.1 reads / million （RPM）的microRNA具有可重复的折叠变化估计（信号到噪声），与多路复用程度无关。检测到的microRNA数量与与microRNA位点对齐的读取数的对数(2)密切相关（R = 0.96）。然而，在测序深度较大的样品中检测到的大多数额外的microrna都在表达范围内，其倍数变化的再现性较低。这些发现阐明了增加多重样本数量与减少测序深度之间的权衡关系，并将有助于设计探索microRNA表达及其在疾病中的作用的大规模临床研究。
Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome. The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However, multiplexing involves a trade-off between increased number of sequenced samples and reduced number of reads per sample (i.e., lower depth of coverage). To assess the effect of different sequencing depths owing to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing one, three, six, nine, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization, replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression >1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the log(2) number of reads aligning to microRNA loci (R = 0.96). However, most additional microRNAs detected in samples with greater sequencing depth were in the range of expression which had lower fold change reproducibility. These findings elucidate the trade-off between increasing the number of samples in a multiplex with decreasing sequencing depth and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.