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

Kellman, B. P.; Baghdassarian, H. M.; Pramparo, T.; Shamie, I.; Gazestani, V. H.; Begzati, A.; Li, S.; Nalabolu, S.; Murray, S.; Lopez, L.; Pierce, K.; Courchesne, E.; Lewis, N. E.

doi:10.1101/2020.04.01.020792

bioRxiv · 10.1101/2020.04.01.020792

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

Kellman, B. P.·Baghdassarian, H. M.·Pramparo, T.·Shamie, I.·Gazestani, V. H.·Begzati, A.·Li, S.·Nalabolu, S.·Murray, S.·Lopez, L.·Pierce, K.·Courchesne, E.·Lewis, N. E.

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Abstract

RNA-Seq is ubiquitous, but depending on the study, sub-optimal sample handling may be required, resulting in repeated freeze-thaw cycles. However, little is known about how each cycle impacts downstream analyses, due to a lack of study and known limitations in common RNA quality metrics, e.g., RIN, at quantifying RNA degradation following repeated freeze-thaws. Here we quantify the impact of repeated freeze-thaw on the reliability of downstream RNA-Seq analysis. To do so, we developed a method to estimate the relative noise between technical replicates independently of RIN. Using this approach we inferred the effect of both RIN and the number of freeze-thaw cycles on sample noise. We find that RIN is unable to fully account for the change in sample noise due to freeze-thaw cycles. Additionally, freeze-thaw is detrimental to sample quality and differential expression (DE) reproducibility, approaching zero after three cycles for poly(A)-enriched samples, wherein the inherent 3 bias in read coverage is more exacerbated by freeze-thaw cycles, while ribosome-depleted samples are less affected by freeze-thaws. 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/020792v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@c2c756org.highwire.dtl.DTLVardef@1ad1c03org.highwire.dtl.DTLVardef@a4136org.highwire.dtl.DTLVardef@13f403f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Kellman, B. P., Baghdassarian, H. M., Pramparo, T., Shamie, I., Gazestani, V. H., Begzati, A., Li, S., Nalabolu, S., Murray, S., Lopez, L., Pierce, K., Courchesne, E., Lewis, N. E.. 2020-04-03. Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing. https://doi.org/10.1101/2020.04.01.020792

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