Poly(a) selection introduces bias and undue noise in direct RNA-sequencing
Abstract Background Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly...
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2022-07-01
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| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12864-022-08762-8 |
| _version_ | 1811287412967473152 |
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| author | Marcus J. Viscardi Joshua A. Arribere |
| author_facet | Marcus J. Viscardi Joshua A. Arribere |
| author_sort | Marcus J. Viscardi |
| collection | DOAJ |
| description | Abstract Background Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species. Results Here, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes’ mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore’s direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality. Conclusions Our work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements. |
| first_indexed | 2024-04-13T03:18:03Z |
| format | Article |
| id | doaj.art-eced616cb6ea40ccadf90300346fc10c |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-04-13T03:18:03Z |
| publishDate | 2022-07-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-eced616cb6ea40ccadf90300346fc10c2022-12-22T03:04:51ZengBMCBMC Genomics1471-21642022-07-0123111010.1186/s12864-022-08762-8Poly(a) selection introduces bias and undue noise in direct RNA-sequencingMarcus J. Viscardi0Joshua A. Arribere1Department of Molecular, Cellular and Developmental Biology, University of California at Santa CruzDepartment of Molecular, Cellular and Developmental Biology, University of California at Santa CruzAbstract Background Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species. Results Here, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes’ mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore’s direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality. Conclusions Our work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements.https://doi.org/10.1186/s12864-022-08762-8Oxford NanoporeDirect RNA-sequencingPoly(a) selectionRNA-sequencingTranscriptomics |
| spellingShingle | Marcus J. Viscardi Joshua A. Arribere Poly(a) selection introduces bias and undue noise in direct RNA-sequencing BMC Genomics Oxford Nanopore Direct RNA-sequencing Poly(a) selection RNA-sequencing Transcriptomics |
| title | Poly(a) selection introduces bias and undue noise in direct RNA-sequencing |
| title_full | Poly(a) selection introduces bias and undue noise in direct RNA-sequencing |
| title_fullStr | Poly(a) selection introduces bias and undue noise in direct RNA-sequencing |
| title_full_unstemmed | Poly(a) selection introduces bias and undue noise in direct RNA-sequencing |
| title_short | Poly(a) selection introduces bias and undue noise in direct RNA-sequencing |
| title_sort | poly a selection introduces bias and undue noise in direct rna sequencing |
| topic | Oxford Nanopore Direct RNA-sequencing Poly(a) selection RNA-sequencing Transcriptomics |
| url | https://doi.org/10.1186/s12864-022-08762-8 |
| work_keys_str_mv | AT marcusjviscardi polyaselectionintroducesbiasandunduenoiseindirectrnasequencing AT joshuaaarribere polyaselectionintroducesbiasandunduenoiseindirectrnasequencing |