Answers.Body: <p>It's possible to do 5' cap enrichment sequencing (e.g. see <a href="https://elifesciences.org/articles/49658#s2-6" rel="nofollow noreferrer">here</a>), and presumably you could look at differences between an enriched vs non-enriched sequencing run to determine whether or not there's a substantial number of sequences with the cap.</p>
  <p>Unfortunately, because the cap is right at the end of the sequence, it zips through a nanopore very quickly, so may be quite difficult to distinguish from any other base at the end. There are also no official models that have been trained to specifically detect the cap, so you'd be initially restricted to looking for signal deviations. If you could find a way to extend the sequence for 20 or so based beyond the cap site, it might be possible to detect it at a single-molecule level.</p>
  <p>Despite all that, this is something that UCSC is working on; see their repository here:</p>
  <p><a href="https://github.com/mitenjain/dRNA_capping_analysis" rel="nofollow noreferrer">https://github.com/mitenjain/dRNA_capping_analysis</a></p>
  
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Answers.CreationDate: 2021-04-28T03:14:10.780Z
Answers.LastActivityDate: 2021-04-28T03:14:10.780Z
Answers.UserDisplayName: gringer
Answers.Body: <p>In short the answer is no, however the 'car-jumping method' described <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC92527/" rel="nofollow noreferrer">here</a> it is possible, but it is about the enzymic MMLV reverse transcription in response to the capping structure. I assume that an alternative reverse transcriptase doesn't do 'car jumping' therefore any sequencing method will pick this up, but the difference between MMLV verse the heat-tolerant style reverse transcriptases. I would assume the secondary structure associated with viral capping prevents a low temperature reverse transcriptase, i.e. MMLV from correctly reproducing the vRNA into cDNA.</p>
  <p>It is important to note that a cap, isn't just a virus thing, its mRNA eukaryotes per se. The cap is a 7-methylguanosine (m7G) linked to the 5′ end of the transcript by a 5′-5′ triphosphate bridge [m7G(5′)ppp(5′)N- or m7G(5′)ppp(5′)Nm in which Nm is a 2′-O methylated nucleoside].</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-11-28T15:27:48.997Z
Answers.LastActivityDate: 2020-11-28T15:36:13.817Z
Answers.LastEditDate: 2020-11-28T15:36:13.817Z
Answers.UserDisplayName: M__
Body: <p>If you sequence a positive-sense  RNA virus genome using NGS via Nanopore, Illumina or even the old Sanger method, is it possible to tell if the virus has 5' (five prime) cap at the 5' end of the RNA genome?</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2023-09-10T02:39:53.983Z
Comments.Text: Hi @LRJoshi, please do consider gringer's answer below. If this addresses your question please do "accept" it.
Comments.UserDisplayName: M__
Tags: <phylogenetics><covid-19><sars-cov-2>
Title: mRNA cap identification by sequencing?
ThreadText: # mRNA cap identification by sequencing?
  
  (Asked by: username_0 on 2020-11-07)
  
  If you sequence a positive-sense RNA virus genome using NGS via Nanopore, Illumina or even the old Sanger method, is it possible to tell if the virus has 5' (five prime) cap at the 5' end of the RNA genome?
  
  **Tags:** phylogenetics, covid-19, sars-cov-2
  
  > **Comments:**
  > **username_1**: Hi @LRJoshi, please do consider username_2's answer below. If this addresses your question please do "accept" it.  
  
  ---
  
  ## 2 answers
  
  ### Answer by username_2 on 2021-04-28. Score: 1
  
  It's possible to do 5' cap enrichment sequencing (e.g. see [here](https://elifesciences.org/articles/49658#s2-6)), and presumably you could look at differences between an enriched vs non-enriched sequencing run to determine whether or not there's a substantial number of sequences with the cap.
  
  Unfortunately, because the cap is right at the end of the sequence, it zips through a nanopore very quickly, so may be quite difficult to distinguish from any other base at the end. There are also no official models that have been trained to specifically detect the cap, so you'd be initially restricted to looking for signal deviations. If you could find a way to extend the sequence for 20 or so based beyond the cap site, it might be possible to detect it at a single-molecule level.
  
  Despite all that, this is something that UCSC is working on; see their repository here:
  
  <https://github.com/mitenjain/dRNA_capping_analysis>
  
  
  ---
  
  ### Answer by username_1 on 2020-11-28. Score: 0
  
  In short the answer is no, however the 'car-jumping method' described [here](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC92527/) it is possible, but it is about the enzymic MMLV reverse transcription in response to the capping structure. I assume that an alternative reverse transcriptase doesn't do 'car jumping' therefore any sequencing method will pick this up, but the difference between MMLV verse the heat-tolerant style reverse transcriptases. I would assume the secondary structure associated with viral capping prevents a low temperature reverse transcriptase, i.e. MMLV from correctly reproducing the vRNA into cDNA.
  
  It is important to note that a cap, isn't just a virus thing, its mRNA eukaryotes per se. The cap is a 7-methylguanosine (m7G) linked to the 5′ end of the transcript by a 5′-5′ triphosphate bridge [m7G(5′)ppp(5′)N- or m7G(5′)ppp(5′)Nm in which Nm is a 2′-O methylated nucleoside].
  
  
  ---
  
  