Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications
ABSTRACTModifications on viral RNAs (vRNAs), either genomic RNAs or RNA transcripts, have complex effects on the viral life cycle and cellular responses to viral infection. The advent of Oxford Nanopore Technologies Direct RNA Sequencing provides a new strategy for studying RNA modifications. To thi...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2024-02-01
|
| Series: | mSystems |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/msystems.01163-23 |
| _version_ | 1797302109099524096 |
|---|---|
| author | Lu Tan Zhihao Guo Xiaoming Wang Dal Young Kim Runsheng Li |
| author_facet | Lu Tan Zhihao Guo Xiaoming Wang Dal Young Kim Runsheng Li |
| author_sort | Lu Tan |
| collection | DOAJ |
| description | ABSTRACTModifications on viral RNAs (vRNAs), either genomic RNAs or RNA transcripts, have complex effects on the viral life cycle and cellular responses to viral infection. The advent of Oxford Nanopore Technologies Direct RNA Sequencing provides a new strategy for studying RNA modifications. To this end, multiple computational tools have been developed, but a systemic evaluation of their performance in mapping vRNA modifications is lacking. Here, 10 computational tools were tested using the Sindbis virus (SINV) RNAs isolated from infected mammalian (BHK-21) or mosquito (C6/36) cells, with in vitro-transcribed RNAs serving as modification-free control. Three single-mode approaches were shown to be inapplicable in the viral context, and three out of seven comparative methods required cutoff adjustments to reduce false-positive predictions. Utilizing optimized cutoffs, an integrated analysis of comparative tools suggested that the intersected predictions of Tombo_com and xPore were significantly enriched compared with the background. Consequently, a pipeline integrating Tombo_com and xPore was proposed for vRNA modification detection; the performance of which was supported by N6-methyladenosine prediction in severe acute respiratory syndrome coronavirus 2 RNAs using publicly available data. When applied to SINV RNAs, this pipeline revealed more intensive modifications in subgenomic RNAs than in genomic RNAs. Modified uridines were frequently identified, exhibiting substantive overlapping between vRNAs generated in different cell lines. On the other hand, the interpretation of other modifications remained unclear, underlining the limitations of the current computational tools despite their notable potential.IMPORTANCEComputational approaches utilizing Oxford Nanopore Technologies Direct RNA Sequencing data were almost exclusively designed to map eukaryotic epitranscriptomes. Therefore, extra caution must be exercised when using these tools to detect vRNA modifications, as in most cases, vRNA modification profiles should be regarded as unknown epitranscriptomes without prior knowledge. Here, we comprehensively evaluated the performance of 10 computational tools in detecting vRNA modification sites. All tested single-mode methods failed to differentiate native and in vitro-transcribed samples. Using optimized cutoff values, seven tested comparative tools generated very different predictions. An integrated analysis showed significant enrichment of Tombo_com and xPore predictions against the background. A pipeline for vRNA modification detection was proposed accordingly and applied to Sindbis virus RNAs. In conclusion, our study underscores the need for the careful application of computational tools to analyze viral epitranscriptomics. It also offers insights into alphaviral RNA modifications, although further validation is required. |
| first_indexed | 2024-03-07T23:31:10Z |
| format | Article |
| id | doaj.art-fd9c36cee03149ba9f79e5a2bc63e93f |
| institution | Directory Open Access Journal |
| issn | 2379-5077 |
| language | English |
| last_indexed | 2024-03-07T23:31:10Z |
| publishDate | 2024-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSystems |
| spelling | doaj.art-fd9c36cee03149ba9f79e5a2bc63e93f2024-02-20T14:00:48ZengAmerican Society for MicrobiologymSystems2379-50772024-02-019210.1128/msystems.01163-23Utilization of nanopore direct RNA sequencing to analyze viral RNA modificationsLu Tan0Zhihao Guo1Xiaoming Wang2Dal Young Kim3Runsheng Li4Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong, ChinaDepartment of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong, ChinaCollege of Veterinary Medicine, Nanjing Agricultural University, Nanjing, ChinaDepartment of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong, ChinaDepartment of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong, ChinaABSTRACTModifications on viral RNAs (vRNAs), either genomic RNAs or RNA transcripts, have complex effects on the viral life cycle and cellular responses to viral infection. The advent of Oxford Nanopore Technologies Direct RNA Sequencing provides a new strategy for studying RNA modifications. To this end, multiple computational tools have been developed, but a systemic evaluation of their performance in mapping vRNA modifications is lacking. Here, 10 computational tools were tested using the Sindbis virus (SINV) RNAs isolated from infected mammalian (BHK-21) or mosquito (C6/36) cells, with in vitro-transcribed RNAs serving as modification-free control. Three single-mode approaches were shown to be inapplicable in the viral context, and three out of seven comparative methods required cutoff adjustments to reduce false-positive predictions. Utilizing optimized cutoffs, an integrated analysis of comparative tools suggested that the intersected predictions of Tombo_com and xPore were significantly enriched compared with the background. Consequently, a pipeline integrating Tombo_com and xPore was proposed for vRNA modification detection; the performance of which was supported by N6-methyladenosine prediction in severe acute respiratory syndrome coronavirus 2 RNAs using publicly available data. When applied to SINV RNAs, this pipeline revealed more intensive modifications in subgenomic RNAs than in genomic RNAs. Modified uridines were frequently identified, exhibiting substantive overlapping between vRNAs generated in different cell lines. On the other hand, the interpretation of other modifications remained unclear, underlining the limitations of the current computational tools despite their notable potential.IMPORTANCEComputational approaches utilizing Oxford Nanopore Technologies Direct RNA Sequencing data were almost exclusively designed to map eukaryotic epitranscriptomes. Therefore, extra caution must be exercised when using these tools to detect vRNA modifications, as in most cases, vRNA modification profiles should be regarded as unknown epitranscriptomes without prior knowledge. Here, we comprehensively evaluated the performance of 10 computational tools in detecting vRNA modification sites. All tested single-mode methods failed to differentiate native and in vitro-transcribed samples. Using optimized cutoff values, seven tested comparative tools generated very different predictions. An integrated analysis showed significant enrichment of Tombo_com and xPore predictions against the background. A pipeline for vRNA modification detection was proposed accordingly and applied to Sindbis virus RNAs. In conclusion, our study underscores the need for the careful application of computational tools to analyze viral epitranscriptomics. It also offers insights into alphaviral RNA modifications, although further validation is required.https://journals.asm.org/doi/10.1128/msystems.01163-23RNA modificationnanoporecomparative methodsalphavirusSindbis virus |
| spellingShingle | Lu Tan Zhihao Guo Xiaoming Wang Dal Young Kim Runsheng Li Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications mSystems RNA modification nanopore comparative methods alphavirus Sindbis virus |
| title | Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications |
| title_full | Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications |
| title_fullStr | Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications |
| title_full_unstemmed | Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications |
| title_short | Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications |
| title_sort | utilization of nanopore direct rna sequencing to analyze viral rna modifications |
| topic | RNA modification nanopore comparative methods alphavirus Sindbis virus |
| url | https://journals.asm.org/doi/10.1128/msystems.01163-23 |
| work_keys_str_mv | AT lutan utilizationofnanoporedirectrnasequencingtoanalyzeviralrnamodifications AT zhihaoguo utilizationofnanoporedirectrnasequencingtoanalyzeviralrnamodifications AT xiaomingwang utilizationofnanoporedirectrnasequencingtoanalyzeviralrnamodifications AT dalyoungkim utilizationofnanoporedirectrnasequencingtoanalyzeviralrnamodifications AT runshengli utilizationofnanoporedirectrnasequencingtoanalyzeviralrnamodifications |