Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing.
Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing.
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DOI：
10.1186/s12864-021-07381-z
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发表时间：
2021-01-21
期刊：
影响因子：
4.4
通讯作者：
Lewis NE
中科院分区：
文献类型：
--
作者：
Kellman BP;Baghdassarian HM;Pramparo T;Shamie I;Gazestani V;Begzati A;Li S;Nalabolu S;Murray S;Lopez L;Pierce K;Courchesne E;Lewis NE
RNA-Seq和样品冻融都是无处不在的。然而，关于冻融对下游分析的影响的知识有限。缺乏对冻融和RNA降解足够敏感的共同质量指标，例如RNA完整性评分，使这种评估具有挑战性。在这里，我们通过检测从自闭症儿童队列中提取的冷冻白细胞中富含聚(A)和核糖体RNA的RNA-SEQ来量化反复冻融循环对RNA-SEQ可靠性的影响。要做到这一点，我们估计相对噪声，或随机计数的百分比，分离技术重复。使用这种方法，我们测量了与RIN和冻融循环相关的噪声。正如预期的那样，RIN没有完全捕获由于冻融而导致的样品降解。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重复性。与核糖体RNA缺失的样品相比，冻融还导致Poly(A)富集型样品沿基因体的阅读覆盖率分布发生了3‘移位，这表明文库制备可能加剧了冻融引起的样品降解。在冰冻组织的文库制备中普遍使用聚(A)-富集法进行RNA测序，因此，在实验设计和数据分析中考虑反复冻融循环对重复性的影响是很重要的。网上版载有补充材料，可在10.1186/s12864-021-07381-z查阅。
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. The online version contains supplementary material available at 10.1186/s12864-021-07381-z.
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DOI：
10.1093/biostatistics/kxr054
发表时间：
2012-04
期刊：
Biostatistics (Oxford, England)
影响因子：
--
作者：
Hansen KD;Irizarry RA;Wu Z
通讯作者：
Wu Z
DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Anders S;Pyl PT;Huber W
通讯作者：
Huber W
影响因子：
7
作者：
Foley, Joseph W.;Zhu, Chunfang;West, Robert B.
通讯作者：
West, Robert B.
影响因子：
25
作者：
Gazestani, Vahid H.;Pramparo, Tiziano;Lewis, Nathan E.
通讯作者：
Lewis, Nathan E.
影响因子：
2.7
作者：
MCGANN, LE;YANG, HY;WALTERSON, M
通讯作者：
WALTERSON, M