Benjamin P Kellman1,2, Hratch M Baghdassarian1,2, Tiziano Pramparo3, Isaac Shamie1,2, Vahid Gazestani1,3, Arjana Begzati4, Shangzhong Li1,5, Srinivasa Nalabolu3, Sarah Murray6, Linda Lopez3, Karen Pierce3, Eric Courchesne3, Nathan E Lewis7,8,9. 1. Department of Pediatrics, University of California, San Diego, USA. 2. Bioinformatics and Systems Biology Program, University of California San Diego, San Diego, USA. 3. Autism Center of Excellence, Department of Neuroscience, University of California San Diego, San Diego, USA. 4. Department of Medicine, University of California San Diego, San Diego, USA. 5. Department of Bioengineering, University of California San Diego, San Diego, USA. 6. Department of Pathology, University of California San Diego, San Diego, USA. 7. Department of Pediatrics, University of California, San Diego, USA. n4lewis@eng.ucsd.edu. 8. Department of Bioengineering, University of California San Diego, San Diego, USA. n4lewis@eng.ucsd.edu. 9. Novo Nordisk Foundation Center for Biosustainability, University of California, San Diego, La Jolla, USA. n4lewis@eng.ucsd.edu.
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.
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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