Journal Article

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

Baghdassarian, Hratch M., Lewis, Nathan E., Courchesne, Eric, Shamie, Isaac, Pierce, Karen, Nalabolu, Srinivasa, Pramparo, Tiziano, Gazestani, Vahid, Murray, Sarah, Lopez, Linda, Kellman, Benjamin P., Li, Shangzhong, Begzati, Arjana

2021

Overview

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. Graphical abstract

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Animal Genetics and Genomics / Autism / Bias / Biological specimens / Biomedical and Life Sciences / Cryopreservation / Cryopreservation of organs, tissues, etc / Data analysis / Datasets / Degradation / Depletion / Design of experiments / Differential expression / Enrichment / Experimental design / Freeze thaw cycles / Freeze-thaw / Freeze-thawing / Freezing / Gene expression / Gene sequencing / Genetic aspects / Genomics / Humans / Leukocytes / Libraries / Life Sciences / Methods / Microarrays / Microbial Genetics and Genomics / Noise / Plant Genetics and Genomics / Polyadenine / Proteomics / Quality control / Reproducibility / Reproducibility of Results / Research Article / Ribonucleic acid / RNA / RNA sequencing / RNA-Seq / rRNA / Sample preparation / Sequence Analysis, RNA / Storage / Thermal properties / Transcriptomic methods