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All posts created by afreise

| posted 26 Jan, 2023 21:19
Hello, I have run into the exact same question for a gene in Usavi_draft_58 (see attached PDF with HHpred and CDD results, good hits to multiple different functions). Was there ever a decision made for this type of gene? The pham has a number of different functions called.
Posted in: Functional AnnotationPham 23485 repA/DNA primase polymerase
| posted 24 Jan, 2023 01:34
Hi phage biologist!

I'm not completely sure what your question is, but I see why this gene was a tricky one. The fact that it is in a pham with only two other draft genes is indeed suspicious. However, like you said there are hits to genes in other phams that do have functions. Extending the start site of your gene will not automatically re-assign the pham, because Phameration is only done once when we auto-annotate the genome. However, the sequence will still change, and it might result in a sequence that does match up well with the genes from those other phams.

First I looked at the pham pages for the two phams that came up a lot as evidence:
https://phagesdb.org/phams/3757/ (this pham contained the hits in HHpred with the best e-values)
https://phagesdb.org/phams/1046/

Since 3757 had the best hits, I went and looked at the pham members. Most of them had a length of 690bp, which happens to be the length of your LORF in PECAAN.

I selected the start site of 48305 in PECAAN, and then went and pulled the sequence that resulted from that start site. I also pulled one sequence from each of the two phams above so I could align and compare them in NCBI BLASTp.

>JulietS_88
MSNKAYHRTHCKVCGAKITDGTQFSTCSQHRRGWNKREVATCPCGQPAENIRSKYCGEACRKKWGKKPPARMVTKICIGCGNEFSRPHYYPGKMKYCSNACSHRQVKKVRDKFIADLPEGAIVFHSGWEIRFWAACLRFDIPIRSYDGPDIETSQGVYRPDFIIGKPNEERVVDVKGWLRPESEVKCREAGVHLVTKQELLRLESGDSLDAHRALLWNSGMNTHTAPLY

>Ava3_101
MSNKAYHRTHCKVCGAKITDGTQFSTCSQHRRGWNKREVATCPCGQPAENIRSKYCGEACRKKWGKKPPARMVTKICIGCGNEFSRPHYYPGKMKYCSNACSHRQVKKVRDKFIADLPEGAIVFHSGWEIRFVAACERFDIPWRSYDGPDIETSQGVYRPDFIIGKPNEERVVDVKGWLRPESEVKCREAGVHLVTKQELLRLESGDSLDAHRALLWNSGMNTHTAPLY

>BananaFence_99
MSICANPECGKEFEQPNKYRTAKTCSKECRYAVSASTTKASSGRWETKVCPCGVEFQSAVNKPKTYHDWDCMMKHRQEDARASRTCENPECGKEFTYFKRQNQRTCSPECRNKVTAMKRENNYPECQTCGVSTGSYNRIYCDEHRPNRPGRKPAPRITATCLCCGEEFTRPENYPGKMKYCSNACSHRQVKKVRDKFIADLPEGAIVFHSMWEVRFVAACERFDIPWRSYDGPDIETSQGVYRPDFIIGKPNEERVVDVKGWLRPESEVKCREAGVHLVTKQELLRLESGDSLDAHRALLWNSGMNTHTAPLY

When I aligned all these sequences in BLAST (try it yourself! use the "align two or more sequences" checkbox), I see an identical match between JulietS_88 and Ava3_101 (the one from Pham 3757). Although the start site we chose doesn't have the best RBS score, it does close the gap quite nicely, and it produces a gene that matches the other pham members.

Using this new sequence, the HHpred hit for 1M0D_B (Endodeoxyribonuclease I; Holliday junction resolvase,) has a probability of 99%, coverage of 36% (just over our threshold), e-value 4^-10, and aligns with residues 2-95 in this PDB entry (https://www.rcsb.org/structure/1M0D). The entire entry is only 140 residues long, so we have a match to most of the protein. This gives me confidence that this is a good hit for this gene, and Holliday junction resolvase can be called as the function.
Posted in: Functional AnnotationJulietS (C1): Holliday junction resolvase?
| posted 24 Jan, 2023 00:21
Hi Debbie,

I looked further into Usavi_26 using the paper you sent. I summarized my results in the attached PDF. Basically, I think Usavi_25 should be NKF for now, and I'm still torn about Usavi_26. The Pfam hit wasn't very good e-value-wise, but I think I'm seeing some potential alignment in secondary structures? I'm not very confident in calling it a TAC at this time given the weakness of this hit, but it IS also two genes upstream of the TMP…

Thanks,
Amanda
Posted in: Cluster B Annotation TipsTail assembly chaperones?
| posted 21 Dec, 2022 18:08
Thank you Debbie!
Posted in: Functional AnnotationBaseplate protein
| posted 20 Dec, 2022 18:17
Hello,

I am annotating phage Emotion (AZ). The gene at stop 12160 (Emotion_draft_16) has some pretty good hits in HHpred to entries that mention baseplate, but I'm not sure if they are sufficient to call anything related to a baseplate. None of the hits mention anything specifically about baseplate J or baseplate wedge, the two currently approved functions. But the hits were strong enough, and apppeared to cover enough of the sequence from Emotion, that I am curious.

I've summarized the data I found in the attachment and also copied some key info below. Any advice on this one?

Emotion_draft_16:
MAEGDFDLNGYKMGLGYDVVVESFDPGSAEWITQDAVAPVGGRRNFGADYASGPTWVLDLATDTHDAASARAALAGAARAWRPSGLATPGYEAILKYTIGGETRRVYGRPRKFAPIANGVSASGVMLATAEFVTSEAFTHAEEARSLTVGLVPVESGGFTSPLISPITTVAGSQRQGLIDDVGGDAPAPMTITIQGPVSGPKVSSSGWYFSLPSLSLAYDQSVTIDTRKGTVRRNDGANLGGLLSRGSTRLVDARLKTGAVEFIYSGTDQTATSTATVTWRPTYHAL

PhagesDB for gene Emotion_draft_16: https://phagesdb.org/genes/Emotion_CDS_16/
HHpred results: https://toolkit.tuebingen.mpg.de/jobs/Emotion_16
Posted in: Functional AnnotationBaseplate protein
| posted 19 Dec, 2022 21:36
As of Dec 2022 I see many calls for a (single) tail assembly chaperone gene in B1 phages, so I assume sufficient supporting evidence has been found to make this call - but I'm not sure what it is? I have attached a screenshot of the HHpred results for Usavi_25.

https://phagesdb.org/genes/Usavi_CDS_25/
Edited 19 Dec, 2022 21:39
Posted in: Cluster B Annotation TipsTail assembly chaperones?
| posted 19 Dec, 2022 20:53
As of Dec 2022, are we leaving off the MuF part, based on this update from Debbie? https://seaphages.org/forums/topic/4628/

So now just: capsid maturation protease?

(I am annotating Usavi (B1). The gene is here: https://phagesdb.org/genes/Usavi_CDS_10/)
Posted in: Cluster B Annotation TipsCapsid maturation protease and MuF-like fusion protein
| posted 28 Oct, 2022 21:15
Hello,

My students are interested in evaluating the presence and number of tRNAs in entire clusters of phages. However, the phagesDB data on tRNAs is often not up to date - it seems like this must be entered manually in a phage's page even after a phage has been finished in Genbank. For example, in Rhodococcus phages, Phamerator suggested that over 40 of the phages had tRNAs, but phagesDB lists almost no phages with tRNAs for that host.

Is this something that could be updated automatically after final annotation is complete? And in the meantime, is there an easy way to pull accurate tRNA numbers/prsence for phages across a cluster and/or host?

Thanks,
Amanda
Posted in: tRNAsData on tRNAs for many phages?
| posted 12 Aug, 2022 16:07
Hi Debbie,

Got it! I figured synteny was the reason the other pham members had been assigned that function.

Thanks,
Amanda
Posted in: Functional AnnotationCluster BE - terminase, small subunit
| posted 11 Aug, 2022 18:53
Hello! I have a follow-up question about another potential small terminase gene, GalacticEye_Draft_6.

I've attached a summary of the analysis I ran based on other forum posts on this topic. Key points below:

Evidence FOR:
    Downstream gene is terminase, large subunit. (Synteny) A few other genes in this pham have called terminase, small subunit (all CS phages); they have e-value of 0 on PhagesDB BLAST. PCOILS appears to predict some coil-coil (I’m not sure if I’m interpreting this correctly DNABIND predicts DNA-binding function

Evidence AGAINST:
    NO HHpred hits of significance for this gene when searching thru PECAAN (SCOPE, PDB, PFam). NO evidence of HTH-binding function
Posted in: Functional AnnotationCluster BE - terminase, small subunit