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Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing
- Source :
- BMC Genomics, BMC genomics, vol 22, iss 1, BMC Genomics, Vol 22, Iss 1, Pp 1-15 (2021)
- Publication Year :
- 2021
- Publisher :
- Springer Science and Business Media LLC, 2021.
-
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. Graphical abstract
- Subjects :
- lcsh:QH426-470
Bioinformatics
lcsh:Biotechnology
Sample (material)
0206 medical engineering
Sample preparation
RNA-Seq
02 engineering and technology
Computational biology
Biology
Medical and Health Sciences
03 medical and health sciences
Differential expression
0302 clinical medicine
Consistency (statistics)
lcsh:TP248.13-248.65
Information and Computing Sciences
Freezing
Genetics
Humans
Frozen tissue
Freeze-thaw
Lead (electronics)
Gene
Collection methods
030304 developmental biology
Cryopreservation
0303 health sciences
Reproducibility
Sequence Analysis, RNA
Reproducibility of Results
Quality control
RNA
Biological Sciences
Ribosomal RNA
Sample quality
lcsh:Genetics
13. Climate action
Degradation (geology)
DNA microarray
Biological system
Sequence Analysis
020602 bioinformatics
030217 neurology & neurosurgery
Research Article
Biotechnology
Subjects
Details
- ISSN :
- 14712164
- Volume :
- 22
- Database :
- OpenAIRE
- Journal :
- BMC Genomics
- Accession number :
- edsair.doi.dedup.....cd8abb7ef130364b83f47dec0df4d465