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

  • Author(s): 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
  • et al.
Abstract

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 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.

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.

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