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| Status |
Public on May 09, 2020 |
| Title |
Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
RNA-Seq is ubiquitous, but depending on the study, sub-optimal sample handling may be required, resulting in repeated freeze-thaw cycles. However, little is known about how each cycle impacts downstream analyses, due to a lack of study and known limitations in common RNA quality metrics, e.g., RIN, at quantifying RNA degradation following repeated freeze-thaws. Here we quantify the impact of repeated freeze-thaw on the reliability of downstream RNA-Seq analysis. To do so, we developed a method to estimate the relative noise between technical replicates independently of RIN. Using this approach we inferred the effect of both RIN and the number of freeze-thaw cycles on sample noise. We find that RIN is unable to fully account for the change in sample noise due to freeze-thaw cycles. Additionally, freeze-thaw is detrimental to sample quality and differential expression (DE) reproducibility, approaching zero after three cycles for poly(A)-enriched samples, wherein the inherent 3’ bias in read coverage is more exacerbated by freeze-thaw cycles, while ribosome-depleted samples are less affected by freeze-thaws. 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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| Overall design |
For 47 samples (from 16 individuals)--and an additional 9 industry standards--, mRNA was extracted using polyA selection with the TruSeq Stranded mRNA library preparation kit (Illumina). Ribosomal depletion was used to prepare an additional 52 samples. Ribosome depletion prepared samples used the TruSeq Stranded Total RNA with RiboZero Gold library preparation kit (Illumina). RNA Integrity Numbers (RIN) were measured using a NanoDrop ND-1000 (ThermoFisher). Poly-A selected samples were sequenced using 50-base pair single end sequencing on a HiSeq4000 (Illumina) to a depth of 25M reads. The ribo-depletion prepared libraries were sequenced using 100-base pair paired end sequencing on a HiSeq4000 (Illumina) to a depth of 50M reads. Fastq files for each sample underwent quality control using FastQC (v0.33). PolyA and adaptor-trimming were conducted using Trimmomatic. Reads were aligned to the gencode annotated (v25) human reference genome (GRCh38) using STAR (v2.4.0). Alignments were processed to sorted SAM files using SAMtools (v1.7). Finally, HTSeq using default settings (v0.6.1) was used to quantify reads.
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| Contributor(s) |
Lewis NE |
| Citation |
Kellman, B.P., Baghdassarian, H.M., Pramparo, T. *et al.* Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing. *BMC Genomics* 22, 69 (2021). https://doi.org/10.1186/s12864-021-07381-z
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| BioProject |
PRJNA627540 |
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| Submission date |
May 08, 2020 |
| Last update date |
Jan 22, 2021 |
| Contact name |
Nathan Lewis |
| E-mail(s) |
n4lewis@eng.ucsd.edu
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| Organization name |
UC San Diego
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| Department |
Pediatrics
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| Street address |
9500 Gilman Dr. MC 0760 BRF2 4a16
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platforms (1) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
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| Samples (108)
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| Relations |
| SRA |
SRP257988 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE150097_polyA_raw_counts.csv.gz |
1.2 Mb |
(ftp)(http) |
CSV |
| GSE150097_read_coverage_distribution.csv.gz |
77.0 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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