Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation.
Depletion
Oxford nanopore technologies
RNA sequencing
Single-cell RNA sequencing
Journal
Biological procedures online
ISSN: 1480-9222
Titre abrégé: Biol Proced Online
Pays: England
ID NLM: 100963717
Informations de publication
Date de publication:
08 Mar 2023
08 Mar 2023
Historique:
received:
05
01
2023
accepted:
14
02
2023
entrez:
8
3
2023
pubmed:
9
3
2023
medline:
9
3
2023
Statut:
epublish
Résumé
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. 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. 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.
Sections du résumé
BACKGROUND
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
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
CONCLUSIONS
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.
Identifiants
pubmed: 36890441
doi: 10.1186/s12575-023-00193-3
pii: 10.1186/s12575-023-00193-3
pmc: PMC9996952
doi:
Types de publication
Journal Article
Langues
eng
Pagination
7Informations de copyright
© 2023. The Author(s).
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