Apr 3, 2020

Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing

BioRxiv : the Preprint Server for Biology
B. P. KellmanNathan E Lewis


RNA-Seq is ubiquitous, but depending on the biological question or the sample source, some RNA-Seq collection methods require variable or sub-optimal sample handling. Rare tissue may need to be fixed or frozen prior to library preparation, or samples may remain at room temperature while other samples or reagents are handled. These challenges create uncharacterized impacts on sample quality. Understanding the relevant experimental factors that impact sample quality is crucial to ensuring reproducible RNA-Seq results. Here, we tested the susceptibility of poly(A)-enriched RNA-Seq results after multiple freeze-thaw cycles. We assessed sample quality independently of RIN by simulating read count variability to quantify the noise between technical replicates. Each additional freeze-thaw cycle increased the random counts between technical replicates by approximately 4%. Additionally, after three freeze-thaw cycles, differential expression reproducibility decreased substantially. The use of poly(A)-enrichment for RNA sequencing is prevalent 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.

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