Evidence map›Paper›PMID 33478392›Full record

ArticleBMC genomics2021

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

Benjamin P Kellman, Hratch M Baghdassarian, Tiziano Pramparo, Isaac Shamie, Vahid Gazestani, Arjana Begzati, Shangzhong Li, Srinivasa Nalabolu, Sarah Murray, Linda Lopez and 3 more

Abstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in BMC genomics, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.

0numbers the graph read from it
0cells of the map it votes in
16citing papers in PubMed
–field-weighted citation impact
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

16 citing papers in PubMed.

  1. Review
  2. Article
  3. Review
  4. Article
  5. EFFICIENT RIBOSOMAL RNA DEPLETION FROMWellcome open research · 2025
    Article
  6. Article
  7. Review
  8. Article
  9. Article
  10. Article
  11. Article
  12. Article
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  16. Review
4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

13 authors.

Benjamin P Kellman *Department of Pediatrics, University of California, San Diego, USA.
Hratch M Baghdassarian *Department of Pediatrics, University of California, San Diego, USA.
Tiziano PramparoAutism Center of Excellence, Department of Neuroscience, University of California San Diego, San Diego, USA.
Isaac ShamieDepartment of Pediatrics, University of California, San Diego, USA.
Vahid GazestaniDepartment of Pediatrics, University of California, San Diego, USA.
Arjana BegzatiDepartment of Medicine, University of California San Diego, San Diego, USA.
Shangzhong LiDepartment of Pediatrics, University of California, San Diego, USA.
Srinivasa NalaboluAutism Center of Excellence, Department of Neuroscience, University of California San Diego, San Diego, USA.
Sarah MurrayDepartment of Pathology, University of California San Diego, San Diego, USA.
Linda LopezAutism Center of Excellence, Department of Neuroscience, University of California San Diego, San Diego, USA.
Karen PierceAutism Center of Excellence, Department of Neuroscience, University of California San Diego, San Diego, USA.
Eric CourchesneAutism Center of Excellence, Department of Neuroscience, University of California San Diego, San Diego, USA.
Nathan E LewisDepartment of Pediatrics, University of California, San Diego, USA. n4lewis@eng.ucsd.edu.ORCID http://orcid.org/0000-0001-7700-3654

Funding

GRADUATE TRAINING PROGRAM IN BIONFORMATICST32GM008806 · NIGMS · UNIVERSITY OF CALIFORNIA, SAN DIEGO · PI BAFNA, VINEET, REN, BING · 2001 to 2020
$6.2M
Unraveling the mammalian secretory pathway through systems biology and algorithm developmentR35GM119850 · NIGMS · UNIVERSITY OF CALIFORNIA, SAN DIEGO · PI LEWIS, NATHAN ENOCH · 2016 to 2025
$4.3M
Developmental Functional Genomics in ASD ToddlersR01MH110558 · NIMH · UNIVERSITY OF CALIFORNIA, SAN DIEGO · PI COURCHESNE, ERIC, LEWIS, NATHAN ENOCH · 2016 to 2020
$3.0M
Discovering Neural Biomarkers of Language and Social Development in ASD ToddlersR01DC016385 · NIDCD · UNIVERSITY OF CALIFORNIA, SAN DIEGO · PI COURCHESNE, ERIC, PIERCE, KAREN L · 2017 to 2021
$3.0M
NIDCD NIH HHS R01 DC016385NIDCD NIH HHS R01-DC016385NIDCD NIH HHS T32GM008806NIGMS NIH HHS R35 GM119850NIGMS NIH HHS T32 GM008806NIMH NIH HHS R01 MH110558NIMH NIH HHS R01-MH110558Novo Nordisk Fonden (DK) NNF10CC1016517R35 GM119850
6 · The paper itself

Abstract

backgroundBoth 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.

resultsHere 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.

conclusionThe 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.

Indexed as

CryopreservationRNAFreezingHumansReproducibility of ResultsSequence Analysis, RNARNADifferential expressionFreeze-thawQuality controlRNA-SeqSample preparation

Identifiers

PMID33478392
PMCPMC7818915

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Registered trials

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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.