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| Status |
Public on Jul 09, 2018 |
| Title |
Systematic assessment of next-generation sequencing for quantitative small RNA profiling: a multiple protocol study across multiple laboratories |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
|
| 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. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.
This SuperSeries is composed of the SubSeries listed below.
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| Overall design |
Nine labs prepared small RNA-seq libraries from 4 common RNA pools: a) 1 equimolar pool of 1,152 synthetic RNAs, b) 2 pools of synthetic RNAs at differing relative concentrations in pool A and B, and c) human plasma RNA pooled from 11 healthy individuals. Each lab prepared libraries from the samples in quadruplicate, using a standardized TruSeq protocol, as well as at least one other protocol of their choice. Refer to individual Series for details.
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| Citation(s) |
30010675 |
| Submission date |
Feb 06, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Muneesh Tewari |
| E-mail(s) |
mtewari@med.umich.edu
|
| Organization name |
University of Michigan
|
| Lab |
4029 BSRB
|
| Street address |
109 Zina Pitcher Pl
|
| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109-2200 |
| Country |
USA |
| |
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| Platforms (7)
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| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
| GPL19424 |
Illumina NextSeq 500 (synthetic construct) |
| GPL19604 |
Illumina HiSeq 2500 (synthetic construct) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
| GPL21440 |
Ion Torrent Proton (synthetic construct) |
| GPL21616 |
Illumina HiSeq 4000 (synthetic construct) |
|
| Samples (149)
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| This SuperSeries is composed of the following SubSeries:
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| GSE94582 |
Systematic assessment of next-generation sequencing for quantitative small RNA profiling: human plasma pool |
| GSE94584 |
Systematic assessment of next-generation sequencing for quantitative small RNA profiling: synthetic equimolar pool |
| GSE94585 |
Systematic assessment of next-generation sequencing for quantitative small RNA profiling: synthetic ratiometric pools |
| GSE108138 |
Systematic assessment of next-generation sequencing for quantitative small RNA profiling: A-to-I editing pools |
|
| Relations |
| BioProject |
PRJNA371516 |
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