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Systematic assessment of next generation sequencing for quantitative small RNA profiling: a multiple protocol study across multiple laboratories

MD Giraldez, RM Spengler, A Etheridge, PM Godoy, AJ Barczak, S Srinivasan, PL De Hoff, K Tanriverdi, A Courtright, S Lu, J Khoory, R Rubio, D Baxter, TAP Driedonks, HPJ Buermans, ENM Nolte-‘t Hoen, H Jiang, K Wang, I Ghiran, Y Wang, K Van Keuren-Jensen, JE Freedman, PG Woodruff, LC Laurent, DJ Erle, DJ Galas, M Tewari
doi: https://doi.org/10.1101/113050
MD Giraldez
1Department of Internal Medicine, University of Michigan
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RM Spengler
1Department of Internal Medicine, University of Michigan
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A Etheridge
5Pacific Northwest Research Institute
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PM Godoy
6Lung Biology Center, Department of Medicine, University of California San Francisco
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AJ Barczak
6Lung Biology Center, Department of Medicine, University of California San Francisco
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S Srinivasan
7University of California, San Diego, Department of Reproductive Medicine and Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA. 92037
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PL De Hoff
7University of California, San Diego, Department of Reproductive Medicine and Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA. 92037
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K Tanriverdi
8University of Massachusetts Medical School, Department of Medicine, Division of Cardiovascular Medicine, Worcester, Massachusetts 01605, USA
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A Courtright
9Neurogenomics, The Translational Genomics Research Institute (TGen), 445 N. Fifth Street, Phoenix, AZ 85004
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S Lu
10Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School
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J Khoory
10Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School
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R Rubio
11Center for Cancer Computational Biology, Dana-Farber Cancer Institute
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D Baxter
12Institute for Systems Biology, 401 Terry Ave North, Seattle, WA 98109
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TAP Driedonks
13Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht, The Netherlands
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HPJ Buermans
14Leiden Genome Technology Center, Department of Human Genetics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands
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ENM Nolte-‘t Hoen
13Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht, The Netherlands
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H Jiang
3Center for Computational Medicine and Bioinformatics, University of Michigan
15Department of Biostatistics, University of Michigan
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K Wang
12Institute for Systems Biology, 401 Terry Ave North, Seattle, WA 98109
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I Ghiran
10Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School
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Y Wang
11Center for Cancer Computational Biology, Dana-Farber Cancer Institute
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K Van Keuren-Jensen
9Neurogenomics, The Translational Genomics Research Institute (TGen), 445 N. Fifth Street, Phoenix, AZ 85004
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JE Freedman
8University of Massachusetts Medical School, Department of Medicine, Division of Cardiovascular Medicine, Worcester, Massachusetts 01605, USA
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PG Woodruff
16Cardiovascular Research Institute and the Department of Medicine, Division of Pulmonary, Critical Care, Sleep, and Allergy, University of California San Francisco
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LC Laurent
7University of California, San Diego, Department of Reproductive Medicine and Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA. 92037
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DJ Erle
6Lung Biology Center, Department of Medicine, University of California San Francisco
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DJ Galas
5Pacific Northwest Research Institute
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M Tewari
1Department of Internal Medicine, University of Michigan
2Department of Biomedical Engineering, University of Michigan
3Center for Computational Medicine and Bioinformatics, University of Michigan
4Biointerfaces Institute, University of Michigan
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Abstract

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.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted May 17, 2017.
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Systematic assessment of next generation sequencing for quantitative small RNA profiling: a multiple protocol study across multiple laboratories
MD Giraldez, RM Spengler, A Etheridge, PM Godoy, AJ Barczak, S Srinivasan, PL De Hoff, K Tanriverdi, A Courtright, S Lu, J Khoory, R Rubio, D Baxter, TAP Driedonks, HPJ Buermans, ENM Nolte-‘t Hoen, H Jiang, K Wang, I Ghiran, Y Wang, K Van Keuren-Jensen, JE Freedman, PG Woodruff, LC Laurent, DJ Erle, DJ Galas, M Tewari
bioRxiv 113050; doi: https://doi.org/10.1101/113050
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Systematic assessment of next generation sequencing for quantitative small RNA profiling: a multiple protocol study across multiple laboratories
MD Giraldez, RM Spengler, A Etheridge, PM Godoy, AJ Barczak, S Srinivasan, PL De Hoff, K Tanriverdi, A Courtright, S Lu, J Khoory, R Rubio, D Baxter, TAP Driedonks, HPJ Buermans, ENM Nolte-‘t Hoen, H Jiang, K Wang, I Ghiran, Y Wang, K Van Keuren-Jensen, JE Freedman, PG Woodruff, LC Laurent, DJ Erle, DJ Galas, M Tewari
bioRxiv 113050; doi: https://doi.org/10.1101/113050

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