Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers
Abstract Background RNA-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 overre...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-07-01
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| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12864-018-4933-1 |
| _version_ | 1823935860668628992 |
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| author | Yu Fu Pei-Hsuan Wu Timothy Beane Phillip D. Zamore Zhiping Weng |
| author_facet | Yu Fu Pei-Hsuan Wu Timothy Beane Phillip D. Zamore Zhiping Weng |
| author_sort | Yu Fu |
| collection | DOAJ |
| description | Abstract Background RNA-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. Results 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. Conclusions 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. |
| first_indexed | 2024-12-16T22:42:59Z |
| format | Article |
| id | doaj.art-98cfcae444e64eac8d2cedd188d8344a |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-16T22:42:59Z |
| publishDate | 2018-07-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-98cfcae444e64eac8d2cedd188d8344a2022-12-21T22:13:23ZengBMCBMC Genomics1471-21642018-07-0119111410.1186/s12864-018-4933-1Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiersYu Fu0Pei-Hsuan Wu1Timothy Beane2Phillip D. Zamore3Zhiping Weng4Bioinformatics Program, Boston UniversityRNA Therapeutics Institute and Howard Hughes Medical Institute, University of Massachusetts Medical SchoolRNA Therapeutics Institute and Howard Hughes Medical Institute, University of Massachusetts Medical SchoolRNA Therapeutics Institute and Howard Hughes Medical Institute, University of Massachusetts Medical SchoolProgram in Bioinformatics and Integrative Biology, University of Massachusetts Medical SchoolAbstract Background RNA-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. Results 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. Conclusions 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.http://link.springer.com/article/10.1186/s12864-018-4933-1RNA-seqSmall RNA-seqUnique molecular identifierUMIPCR duplicatesPCR cycle |
| spellingShingle | Yu Fu Pei-Hsuan Wu Timothy Beane Phillip D. Zamore Zhiping Weng Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers BMC Genomics RNA-seq Small RNA-seq Unique molecular identifier UMI PCR duplicates PCR cycle |
| title | Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers |
| title_full | Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers |
| title_fullStr | Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers |
| title_full_unstemmed | Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers |
| title_short | Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers |
| title_sort | elimination of pcr duplicates in rna seq and small rna seq using unique molecular identifiers |
| topic | RNA-seq Small RNA-seq Unique molecular identifier UMI PCR duplicates PCR cycle |
| url | http://link.springer.com/article/10.1186/s12864-018-4933-1 |
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