Kellman Benjamin P, Baghdassarian Hratch M, Pramparo Tiziano, Shamie Isaac, Gazestani Vahid, Begzati Arjana, Li Shangzhong, Nalabolu Srinivasa, Murray Sarah, Lopez Linda, Pierce Karen, Courchesne Eric, Lewis Nathan E
Department of Pediatrics, University of California, San Diego, USA.
Bioinformatics and Systems Biology Program, University of California San Diego, San Diego, USA.
BMC Genomics. 2021 Jan 21;22(1):69. doi: 10.1186/s12864-021-07381-z.
Both RNA-Seq and sample freeze-thaw are ubiquitous. However, knowledge about the impact of freeze-thaw on downstream analyses is limited. The lack of common quality metrics that are sufficiently sensitive to freeze-thaw and RNA degradation, e.g. the RNA Integrity Score, makes such assessments challenging.
Here we quantify the impact of repeated freeze-thaw cycles on the reliability of RNA-Seq by examining poly(A)-enriched and ribosomal RNA depleted RNA-seq from frozen leukocytes drawn from a toddler Autism cohort. To do so, we estimate the relative noise, or percentage of random counts, separating technical replicates. Using this approach we measured noise associated with RIN and freeze-thaw cycles. As expected, RIN does not fully capture sample degradation due to freeze-thaw. We further examined differential expression results and found that three freeze-thaws should extinguish the differential expression reproducibility of similar experiments. Freeze-thaw also resulted in a 3' shift in the read coverage distribution along the gene body of poly(A)-enriched samples compared to ribosomal RNA depleted samples, suggesting that library preparation may exacerbate freeze-thaw-induced sample degradation.
The use of poly(A)-enrichment for RNA sequencing is pervasive in library preparation of frozen tissue, and thus, it is important during experimental design and data analysis to consider the impact of repeated freeze-thaw cycles on reproducibility.
RNA测序（RNA-Seq）和样本冻融都很常见。然而，关于冻融对下游分析影响的了解有限。缺乏对冻融和RNA降解足够敏感的通用质量指标，例如RNA完整性评分，使得此类评估具有挑战性。
在这里，我们通过检查来自一名幼儿自闭症队列的冷冻白细胞的聚腺苷酸（poly(A)）富集和核糖体RNA去除的RNA测序，量化了重复冻融循环对RNA-Seq可靠性的影响。为此，我们估计了分离技术重复样本的相对噪声或随机计数百分比。使用这种方法，我们测量了与RNA完整性评分（RIN）和冻融循环相关的噪声。正如预期的那样，RIN不能完全捕捉到由于冻融导致的样本降解。我们进一步检查了差异表达结果，发现三次冻融应该会消除类似实验的差异表达可重复性。与核糖体RNA去除的样本相比，冻融还导致聚腺苷酸富集样本沿基因体的读取覆盖分布发生3'偏移，这表明文库制备可能会加剧冻融诱导的样本降解。
在冷冻组织的文库制备中，使用聚腺苷酸富集进行RNA测序很普遍，因此，在实验设计和数据分析过程中，考虑重复冻融循环对可重复性的影响很重要。
