Comparison of rRNA depletion methods for efficient bacterial mRNA sequencing
Abstract Current methods of high-throughput RNA sequencing of prokaryotes, including transcriptome analysis or ribosomal profiling, need deep sequencing to achieve sufficient numbers of effective reads (e.g., mapping to mRNA) in order to also find weakly expressed genetic elements. The fraction of h...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-09710-y |
| _version_ | 1819011610746814464 |
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| author | Anika Wahl Christopher Huptas Klaus Neuhaus |
| author_facet | Anika Wahl Christopher Huptas Klaus Neuhaus |
| author_sort | Anika Wahl |
| collection | DOAJ |
| description | Abstract Current methods of high-throughput RNA sequencing of prokaryotes, including transcriptome analysis or ribosomal profiling, need deep sequencing to achieve sufficient numbers of effective reads (e.g., mapping to mRNA) in order to also find weakly expressed genetic elements. The fraction of high-quality reads mapping to coding RNAs (i.e., mRNA) is mainly influenced by the large content of rRNA and, to a lesser extent, tRNA in total RNA. Thus, depletion of rRNA increases coverage and thus sequencing costs. RiboZero, a depletion kit based on probe hybridisation and rRNA-removal was found to be most efficient in the past, but it was discontinued in 2018. To facilitate comparability with previous experiments and to help choose adequate replacements, we compare three commercially available rRNA depletion kits also based on hybridization and magnetic beads, i.e., riboPOOLs, RiboMinus and MICROBExpress, with the former RiboZero. Additionally, we constructed biotinylated probes for magnetic bead capture and rRNA depletion in this study. Based on E. coli, we found similar efficiencies in rRNA depletion for riboPOOLs and the self-made depletion method; both comparable to the former RiboZero, followed by RiboMinus, succeeded by MICROBExpress. Further, our in-house protocol allows customized species-specific rRNA or even tRNA depletion or depletion of other RNA targets. Both, the self-made biotinylated probes and riboPOOLs, were most successful in reducing the rRNA content and thereby increasing sequencing depth concerning mRNA reads. Additionally, the number of reads matching to weakly expressed genes are increased. In conclusion, the self-made specific biotinylated probes and riboPOOLs are an adequate replacement for the former RiboZero. Both are very efficient in depleting rRNAs, increasing mRNA reads and thus sequencing efficiency. |
| first_indexed | 2024-12-21T01:30:54Z |
| format | Article |
| id | doaj.art-d85b100d4a1e417a825a7ad6eb2aee40 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-21T01:30:54Z |
| publishDate | 2022-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-d85b100d4a1e417a825a7ad6eb2aee402022-12-21T19:20:22ZengNature PortfolioScientific Reports2045-23222022-04-0112111110.1038/s41598-022-09710-yComparison of rRNA depletion methods for efficient bacterial mRNA sequencingAnika Wahl0Christopher Huptas1Klaus Neuhaus2Core Facility Microbiome, ZIEL – Institute for Food and Health, Technische Universität MünchenChair for Microbial Ecology, Technische Universität MünchenCore Facility Microbiome, ZIEL – Institute for Food and Health, Technische Universität MünchenAbstract Current methods of high-throughput RNA sequencing of prokaryotes, including transcriptome analysis or ribosomal profiling, need deep sequencing to achieve sufficient numbers of effective reads (e.g., mapping to mRNA) in order to also find weakly expressed genetic elements. The fraction of high-quality reads mapping to coding RNAs (i.e., mRNA) is mainly influenced by the large content of rRNA and, to a lesser extent, tRNA in total RNA. Thus, depletion of rRNA increases coverage and thus sequencing costs. RiboZero, a depletion kit based on probe hybridisation and rRNA-removal was found to be most efficient in the past, but it was discontinued in 2018. To facilitate comparability with previous experiments and to help choose adequate replacements, we compare three commercially available rRNA depletion kits also based on hybridization and magnetic beads, i.e., riboPOOLs, RiboMinus and MICROBExpress, with the former RiboZero. Additionally, we constructed biotinylated probes for magnetic bead capture and rRNA depletion in this study. Based on E. coli, we found similar efficiencies in rRNA depletion for riboPOOLs and the self-made depletion method; both comparable to the former RiboZero, followed by RiboMinus, succeeded by MICROBExpress. Further, our in-house protocol allows customized species-specific rRNA or even tRNA depletion or depletion of other RNA targets. Both, the self-made biotinylated probes and riboPOOLs, were most successful in reducing the rRNA content and thereby increasing sequencing depth concerning mRNA reads. Additionally, the number of reads matching to weakly expressed genes are increased. In conclusion, the self-made specific biotinylated probes and riboPOOLs are an adequate replacement for the former RiboZero. Both are very efficient in depleting rRNAs, increasing mRNA reads and thus sequencing efficiency.https://doi.org/10.1038/s41598-022-09710-y |
| spellingShingle | Anika Wahl Christopher Huptas Klaus Neuhaus Comparison of rRNA depletion methods for efficient bacterial mRNA sequencing Scientific Reports |
| title | Comparison of rRNA depletion methods for efficient bacterial mRNA sequencing |
| title_full | Comparison of rRNA depletion methods for efficient bacterial mRNA sequencing |
| title_fullStr | Comparison of rRNA depletion methods for efficient bacterial mRNA sequencing |
| title_full_unstemmed | Comparison of rRNA depletion methods for efficient bacterial mRNA sequencing |
| title_short | Comparison of rRNA depletion methods for efficient bacterial mRNA sequencing |
| title_sort | comparison of rrna depletion methods for efficient bacterial mrna sequencing |
| url | https://doi.org/10.1038/s41598-022-09710-y |
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