Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers [preprint]
UMass Chan AffiliationsDepartment of Biochemistry and Molecular Pharmacology
RNA Therapeutics Institute
Program in Bioinformatics and Integrative Biology
Keywordspolymerase chain reaction
Unique molecular identifiers
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AbstractRNA-seq and small RNA-seq are powerful, quantitative tools to study gene regulation and function. Common high-throughput sequencing methods rely on polymerase chain reaction (PCR) to expand the starting material, but not every molecule amplifies equally, causing some to be overrepresented. Unique molecular identifiers (UMIs) can be used to distinguish undesirable PCR duplicates derived from a single molecule and identical but biologically meaningful reads from different molecules. We have incorporated UMIs into RNA-seq and small RNA-seq protocols and developed tools to analyze the resulting data. Our UMIs contain stretches of random nucleotides whose lengths sufficiently capture diverse molecule species in both RNA-seq and small RNA-seq libraries generated from mouse testis. Our approach yields high-quality data while allowing unique tagging of all molecules in high-depth libraries. Using simulated and real datasets, we demonstrate that our methods increase the reproducibility of RNA-seq and small RNA-seq data. Notably, we find that the amount of starting material and sequencing depth, but not the number of PCR cycles, determine PCR duplicate frequency. Finally, we show that computational removal of PCR duplicates based only on their mapping coordinates introduces substantial bias into data analysis.
bioRxiv 251892; doi: https://doi.org/10.1101/251892. Link to preprint on bioRxiv service.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/29275
Now published in BMC Genomics, doi: 10.1186/s12864-018-4933-1
RightsThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Except where otherwise noted, this item's license is described as The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.