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Research article
Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing
Nathan Lewis
UCSD
Corresponding Author
ORCiD: https://orcid.org/0000-0001-7700-3654
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
RNA-Seq, quality control, freeze-thaw, sample preparation, differential expression
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Received 22 Sep, 2020
Reviewer #4 agreed
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Reviewer #1 agreed
On 16 Sep, 2020
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On 16 Sep, 2020
Reviewer #3 agreed
On 16 Sep, 2020
Reviewers invited
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Subject Areas
Epigenetics & Genomics
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This preprint is under consideration at BMC Genomics. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing
Nathan Lewis
UCSD
Corresponding Author
ORCiD: https://orcid.org/0000-0001-7700-3654
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 3
Posted 12 Jan, 2021
No community comments so far
Submission checks complete
On 31 Dec, 2020
No comments provided
Editorial decision: Minor revision
On 27 Dec, 2020
Review #3 received
Received 24 Dec, 2020
Reviewer #3 agreed
On 06 Dec, 2020
Reviewer #2 agreed
On 01 Dec, 2020
Reviewers invited
Invitations sent on 01 Dec, 2020
Reviewer #1 agreed
On 01 Dec, 2020
Review #1 received
Received 01 Dec, 2020
Review #2 received
Received 01 Dec, 2020
Editor assigned
On 09 Nov, 2020
Submission checks complete
On 09 Nov, 2020
Editor invited
On 09 Nov, 2020
No comments provided
Review #2 received
Received 08 Oct, 2020
Editorial decision: Major revision
On 08 Oct, 2020
Review #1 received
Received 02 Oct, 2020
Review #3 received
Received 22 Sep, 2020
Review #4 received
Received 22 Sep, 2020
Reviewer #4 agreed
On 20 Sep, 2020
Reviewer #1 agreed
On 16 Sep, 2020
Reviewer #2 agreed
On 16 Sep, 2020
Reviewer #3 agreed
On 16 Sep, 2020
Reviewers invited
Invitations sent on 15 Sep, 2020
First submitted
On 25 Aug, 2020
Editor assigned
On 25 Aug, 2020
Submission checks complete
On 24 Aug, 2020
Editor invited
On 24 Aug, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Epigenetics & Genomics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6