SEA-PHAGES | Finding introns

Link to this post \| posted 26 Jun, 2019 18:52
welkin	From Amy Sprenkle: "We from cohort 11 adopted a Cluster J phage in the spring of 2019 to make sure we really got trained in all of the bioinformatics tools available. Hannaconda did not disappoint. I relied heavily on the Pope J Cluster paper for guidance, and worked on the TAC frameshift successfully from the excellent bioinformatics guide and help from Welkin. We used PECAAN and had two tRNAs called, and I learned at the symposium that the web-based aragorn would call introns as well. I failed to throughly explore this tool before submitting the genome, so I will continue to work on that. I message in this string because when searching for J cluster and introns, this is what pops up. It is not at all clear to me how one uses phamerator to see the introns, because they are not described in phamerator the way they are in the paper. Also, some of the J phage from the paper have an intron and others do not. Those that do are not consistent with regard to their association with HNH endonucleases. Any help with better calling of an intron would be appreciated."

Link to this post | posted 26 Jun, 2019 18:52

From Amy Sprenkle:
"We from cohort 11 adopted a Cluster J phage in the spring of 2019 to make sure we really got trained in all of the bioinformatics tools available. Hannaconda did not disappoint. I relied heavily on the Pope J Cluster paper for guidance, and worked on the TAC frameshift successfully from the excellent bioinformatics guide and help from Welkin. We used PECAAN and had two tRNAs called, and I learned at the symposium that the web-based aragorn would call introns as well. I failed to throughly explore this tool before submitting the genome, so I will continue to work on that. I message in this string because when searching for J cluster and introns, this is what pops up. It is not at all clear to me how one uses phamerator to see the introns, because they are not described in phamerator the way they are in the paper. Also, some of the J phage from the paper have an intron and others do not. Those that do are not consistent with regard to their association with HNH endonucleases.
Any help with better calling of an intron would be appreciated."

Link to this post \| posted 01 Oct, 2019 20:57
asprenkle	I noticed this request for help in calling introns had received no reply, and Hannaconda is still awaiting QC, whereas our other phage from the Genome exchange has received an accession number. Is there anything I should be doing to address this?

Link to this post \| posted 03 Oct, 2019 15:57
debbie	Amy, I am the one QCing this genome and I have not gotten to it yet. Welkin, would you respond to the intron question? I think that info that may be helpful is found here https://seaphages.org/forums/topic/4926/?page=1#post-7336 Thanks, debbie Edited 03 Oct, 2019 17:06

Link to this post \| posted 04 Oct, 2019 13:38
welkin	Hi Amy, The only real way to tell if you have an intron is at the bench, so for the most part we do not add them to annotations. The exception being cluster J, as you noted above, where we have bench data for one in the capsid gene of LittleE and one in a minor tail gene in BAKA. So if your phages is identical in sequence to one of the two that we've demonstrated are present at the bench, you should add them. The comment about using Phamerator to find them only really works if an essential gene is split into two pieces in one phage where it is only present in one piece in the close relatives. This is how we discovered that LittleE and BAKA had introns in the first place— we knew that the capsid gene should only be one gene. So to use Phamerator, you'd need to know that genes are essential and should be only one piece— which doesn't apply to most of the phage genes as we do not know what most of them do. If you have an intron identical to one of the previously characterized ones, you can use the nucleotide sequences in Phamerator to show you exactly where the boundaries are— just get the nucleotide sequence of each gene in question and then align them against each other using BLAST.

Link to this post | posted 04 Oct, 2019 13:38

welkin

Hi Amy,
The only real way to tell if you have an intron is at the bench, so for the most part we do not add them to annotations.
The exception being cluster J, as you noted above, where we have bench data for one in the capsid gene of LittleE and one in a minor tail gene in BAKA. So if your phages is identical in sequence to one of the two that we've demonstrated are present at the bench, you should add them.

The comment about using Phamerator to find them only really works if an essential gene is split into two pieces in one phage where it is only present in one piece in the close relatives. This is how we discovered that LittleE and BAKA had introns in the first place— we knew that the capsid gene should only be one gene. So to use Phamerator, you'd need to know that genes are essential and should be only one piece— which doesn't apply to most of the phage genes as we do not know what most of them do.
If you have an intron identical to one of the previously characterized ones, you can use the nucleotide sequences in Phamerator to show you exactly where the boundaries are— just get the nucleotide sequence of each gene in question and then align them against each other using BLAST.

Link to this post \| posted 04 Oct, 2019 17:28
asprenkle	Thank you so much, Debbie and Welkin! I know you have so much to do. I was noticing on the phagesdb page for Hannaconda, it is not in the archives, which might lower any enthusiasm to get it out of the genome exchange and publish the sequence anyway. It was such a good exercise for us as newbies with no genome of our own though, and I'm glad we tried to tackle it. I will keep focused on meeting the goal this second semester to get enough phage of our own to archive and sequence!

Link to this post | posted 04 Oct, 2019 17:28

asprenkle

Thank you so much, Debbie and Welkin! I know you have so much to do. I was noticing on the phagesdb page for Hannaconda, it is not in the archives, which might lower any enthusiasm to get it out of the genome exchange and publish the sequence anyway. It was such a good exercise for us as newbies with no genome of our own though, and I'm glad we tried to tackle it.
I will keep focused on meeting the goal this second semester to get enough phage of our own to archive and sequence!

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Finding introns