Background: 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.Results: 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

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... tes 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.Conclusion: 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.