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| Status |
Public on Jul 09, 2018 |
| Title |
Systematic assessment of next-generation sequencing for quantitative small RNA profiling: synthetic ratiometric pools |
| Organism |
synthetic construct |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
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| Summary |
Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. Using common sets of reference samples, we evaluated the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. As part of this larger study, we assessed reproducibility and accuracy of relative expression measurements using two pools of synthetic small RNA sequences, where subsets of the sRNAs vary in relative amount between pools A and B. The pools each contain 334 small RNAs, varying by 15 different ratios between pools A and B (10:1, 8:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4 1:5, 1:8, 1:10). We find that although the sequencing bias varies extensively between protocols, the relative abundance measured between samples A and B is largely reproducible and accurate across labs and protocols. These results suggest that measurements of differential expression should be comparable across institutions and library preparation technologies.
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| Overall design |
Two pools of synthetic RNAs were sequenced by 9 different labs, using 1 common library construction protocol (TruSeq), and at least one additional protocol of their choice. The RNA pools contain a common set of synthetic oligos at differing relative concentrations in the SynthA and SynthB pools. A total of 40 sample libraries was produced (20 each for SynthA and SynthB), each in quadruplicate.
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| Contributor(s) |
Tewari M |
| Citation(s) |
30010675 |
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| Submission date |
Feb 06, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Muneesh Tewari |
| E-mail(s) |
mtewari@med.umich.edu
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| Organization name |
University of Michigan
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| Lab |
4029 BSRB
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| Street address |
109 Zina Pitcher Pl
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109-2200 |
| Country |
USA |
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| Platforms (4)
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| GPL19424 |
Illumina NextSeq 500 (synthetic construct) |
| GPL19604 |
Illumina HiSeq 2500 (synthetic construct) |
| GPL21440 |
Ion Torrent Proton (synthetic construct) |
| GPL21616 |
Illumina HiSeq 4000 (synthetic construct) |
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| Samples (50)
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| This SubSeries is part of SuperSeries: |
| GSE94586 |
Systematic assessment of next-generation sequencing for quantitative small RNA profiling: a multiple protocol study across multiple laboratories |
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| Relations |
| BioProject |
PRJNA371517 |
| SRA |
SRP098949 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE94585_RAW.tar |
1.4 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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