Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation
Abstract Background RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundan...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-03-01
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| Series: | Biological Procedures Online |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12575-023-00193-3 |
| _version_ | 1797865539676143616 |
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| author | Celine Everaert Jasper Verwilt Kimberly Verniers Niels Vandamme Alvaro Marcos Rubio Jo Vandesompele Pieter Mestdagh |
| author_facet | Celine Everaert Jasper Verwilt Kimberly Verniers Niels Vandamme Alvaro Marcos Rubio Jo Vandesompele Pieter Mestdagh |
| author_sort | Celine Everaert |
| collection | DOAJ |
| description | Abstract Background RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species. Results We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3′ end sequencing and long-read sequencing, and MALAT1 in single-cell 3′ end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity. Conclusion Our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol. |
| first_indexed | 2024-04-09T23:10:49Z |
| format | Article |
| id | doaj.art-72d160ee571d4042a249a3fb72af33c7 |
| institution | Directory Open Access Journal |
| issn | 1480-9222 |
| language | English |
| last_indexed | 2024-04-09T23:10:49Z |
| publishDate | 2023-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Biological Procedures Online |
| spelling | doaj.art-72d160ee571d4042a249a3fb72af33c72023-03-22T10:26:05ZengBMCBiological Procedures Online1480-92222023-03-0125111910.1186/s12575-023-00193-3Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library PreparationCeline Everaert0Jasper Verwilt1Kimberly Verniers2Niels Vandamme3Alvaro Marcos Rubio4Jo Vandesompele5Pieter Mestdagh6Department of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityCancer Research Institute Ghent, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityAbstract Background RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species. Results We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3′ end sequencing and long-read sequencing, and MALAT1 in single-cell 3′ end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity. Conclusion Our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.https://doi.org/10.1186/s12575-023-00193-3RNA sequencingOxford nanopore technologiesSingle-cell RNA sequencingDepletion |
| spellingShingle | Celine Everaert Jasper Verwilt Kimberly Verniers Niels Vandamme Alvaro Marcos Rubio Jo Vandesompele Pieter Mestdagh Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation Biological Procedures Online RNA sequencing Oxford nanopore technologies Single-cell RNA sequencing Depletion |
| title | Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation |
| title_full | Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation |
| title_fullStr | Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation |
| title_full_unstemmed | Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation |
| title_short | Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation |
| title_sort | blocking abundant rna transcripts by high affinity oligonucleotides during transcriptome library preparation |
| topic | RNA sequencing Oxford nanopore technologies Single-cell RNA sequencing Depletion |
| url | https://doi.org/10.1186/s12575-023-00193-3 |
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