Short paired-end reads trump long single-end reads for expression analysis
Abstract Background Typical experimental design advice for expression analyses using RNA-seq generally assumes that single-end reads provide robust gene-level expression estimates in a cost-effective manner, and that the additional benefits obtained from paired-end sequencing are not worth the addit...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2020-04-01
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| Series: | BMC Bioinformatics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12859-020-3484-z |
| _version_ | 1818881309373628416 |
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| author | Adam H. Freedman John M. Gaspar Timothy B. Sackton |
| author_facet | Adam H. Freedman John M. Gaspar Timothy B. Sackton |
| author_sort | Adam H. Freedman |
| collection | DOAJ |
| description | Abstract Background Typical experimental design advice for expression analyses using RNA-seq generally assumes that single-end reads provide robust gene-level expression estimates in a cost-effective manner, and that the additional benefits obtained from paired-end sequencing are not worth the additional cost. However, in many cases (e.g., with Illumina NextSeq and NovaSeq instruments), shorter paired-end reads and longer single-end reads can be generated for the same cost, and it is not obvious which strategy should be preferred. Using publicly available data, we test whether short-paired end reads can achieve more robust expression estimates and differential expression results than single-end reads of approximately the same total number of sequenced bases. Results At both the transcript and gene levels, 2 × 40 paired-end reads unequivocally provide expression estimates that are more highly correlated with 2 × 125 than 1 × 75 reads; in nearly all cases, those correlations are also greater than for 1 × 125, despite the greater total number of sequenced bases for the latter. Across an array of metrics, differential expression tests based upon 2 × 40 consistently outperform those using 1 × 75. Conclusion Researchers seeking a cost-effective approach for gene-level expression analysis should prefer short paired-end reads over a longer single-end strategy. Short paired-end reads will also give reasonably robust expression estimates and differential expression results at the isoform level. |
| first_indexed | 2024-12-19T14:59:49Z |
| format | Article |
| id | doaj.art-ec4b6f7fa9644b899767bee2b0221432 |
| institution | Directory Open Access Journal |
| issn | 1471-2105 |
| language | English |
| last_indexed | 2024-12-19T14:59:49Z |
| publishDate | 2020-04-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Bioinformatics |
| spelling | doaj.art-ec4b6f7fa9644b899767bee2b02214322022-12-21T20:16:36ZengBMCBMC Bioinformatics1471-21052020-04-0121111110.1186/s12859-020-3484-zShort paired-end reads trump long single-end reads for expression analysisAdam H. Freedman0John M. Gaspar1Timothy B. Sackton2Informatics Group, Harvard UniversityInformatics Group, Harvard UniversityInformatics Group, Harvard UniversityAbstract Background Typical experimental design advice for expression analyses using RNA-seq generally assumes that single-end reads provide robust gene-level expression estimates in a cost-effective manner, and that the additional benefits obtained from paired-end sequencing are not worth the additional cost. However, in many cases (e.g., with Illumina NextSeq and NovaSeq instruments), shorter paired-end reads and longer single-end reads can be generated for the same cost, and it is not obvious which strategy should be preferred. Using publicly available data, we test whether short-paired end reads can achieve more robust expression estimates and differential expression results than single-end reads of approximately the same total number of sequenced bases. Results At both the transcript and gene levels, 2 × 40 paired-end reads unequivocally provide expression estimates that are more highly correlated with 2 × 125 than 1 × 75 reads; in nearly all cases, those correlations are also greater than for 1 × 125, despite the greater total number of sequenced bases for the latter. Across an array of metrics, differential expression tests based upon 2 × 40 consistently outperform those using 1 × 75. Conclusion Researchers seeking a cost-effective approach for gene-level expression analysis should prefer short paired-end reads over a longer single-end strategy. Short paired-end reads will also give reasonably robust expression estimates and differential expression results at the isoform level.http://link.springer.com/article/10.1186/s12859-020-3484-zRNA-seqShort read sequencingDifferential expression |
| spellingShingle | Adam H. Freedman John M. Gaspar Timothy B. Sackton Short paired-end reads trump long single-end reads for expression analysis BMC Bioinformatics RNA-seq Short read sequencing Differential expression |
| title | Short paired-end reads trump long single-end reads for expression analysis |
| title_full | Short paired-end reads trump long single-end reads for expression analysis |
| title_fullStr | Short paired-end reads trump long single-end reads for expression analysis |
| title_full_unstemmed | Short paired-end reads trump long single-end reads for expression analysis |
| title_short | Short paired-end reads trump long single-end reads for expression analysis |
| title_sort | short paired end reads trump long single end reads for expression analysis |
| topic | RNA-seq Short read sequencing Differential expression |
| url | http://link.springer.com/article/10.1186/s12859-020-3484-z |
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