PDF(Pubmed)
Abstract：
We present an easy-to-reproduce manual miniaturized full-length RNA sequencing (RNAseq) library preparation workflow that does not require the upfront investment in expensive lab equipment or long setup times. With minimal adjustments to an established commercial protocol, we were able to manually miniaturize the RNAseq library preparation by a factor of up to 1:8. This led to cost savings for miniaturized library preparation of up to 86.1% compared to the gold standard. The resulting data were the basis of a rigorous quality control analysis that inspected: sequencing quality metrics, gene body coverage, raw read duplications, alignment statistics, read pair duplications, detected transcripts and sequence variants. We also included a deep dive data analysis identifying rRNA contamination and suggested ways to circumvent these. In the end, we could not find any indication of biases or inaccuracies caused by the RNAseq library miniaturization. The variance in detected transcripts was minimal and not influenced by the miniaturization level. Our results suggest that the workflow is highly reproducible and the sequence data suitable for downstream analyses such as differential gene expression analysis or variant calling.
摘要：
我们提出了一种易于复制的手动小型化全长RNA测序（RNAseq）文库制备工作流程，不需要昂贵的实验室设备或长时间的前期投资。只需对已建立的商业协议进行最小的调整，我们能够手动小型化RNAseq文库制备高达1:8的因子。与黄金标准相比，这导致小型化文库制备的成本节省高达86.1％。所得数据是严格的质量控制分析的基础，该分析检查了：测序质量指标，基因体覆盖，原始读取重复，对齐统计，读取对重复，检测到转录本和序列变异。我们还进行了深入的数据分析，确定了rRNA污染，并提出了规避这些污染的方法。最后,我们没有发现任何由RNAseq文库小型化引起的偏差或不准确的迹象。检测到的转录物的方差最小，不受小型化水平的影响。我们的结果表明，工作流程是高度可重复的，序列数据适用于下游分析，如差异基因表达分析或变异识别。
