SEA-PHAGES | Tail Assembly Chaperone

Link to this post \| posted 02 Mar, 2020 01:48
stpage	Not annotating frameshift in absence of wet bench evidence, GC-rich region but not obvious slippery sequence. Some in cluster have two tail assembly chaperone back to back in same frame (Sour, NHagos), most overlapping in separate frames (Cloverminnie, Mariokart).

Link to this post \| posted 02 Mar, 2020 14:36
welkin	Hi Shallee– sounds like a good decision to me.

Link to this post \| posted 02 Mar, 2020 19:09
erin.windsor	Hi Shallee, NHagos annotator here. NHagos and Sour tail assembly chaperones are not in the same frame. They were annotated with frameshifts so it looks back-to-back in phamerator. You cannot tell which frame it is in from phamerator. Download the NHagos minimal file to see our frameshift. You can also look up the genbank entry to see that is was annotated at nucleotide 17143. There are 4 G's in a row. Sour also had these 4 G's in a row. Our complete notes file does not have the frameshift annotated.

Link to this post | posted 02 Mar, 2020 19:09

erin.windsor

Hi Shallee,

NHagos annotator here. NHagos and Sour tail assembly chaperones are not in the same frame. They were annotated with frameshifts so it looks back-to-back in phamerator. You cannot tell which frame it is in from phamerator. Download the NHagos minimal file to see our frameshift. You can also look up the genbank entry to see that is was annotated at nucleotide 17143. There are 4 G's in a row. Sour also had these 4 G's in a row. Our complete notes file does not have the frameshift annotated.

Link to this post \| posted 03 Mar, 2020 17:15
stpage	Thanks again, Erin. It makes more sense looking at the GenBank entry rather than Phamerator. I don't have that many Gs, but I'll try a mockup of the frameshift and see how it looks in Mariokart.

Link to this post \| posted 03 Mar, 2020 17:50
debbie	Hi all, I am going to chime in here, but my understanding today is that every phage may not have the G/T programmed frameshift. In lots of cases we can only find homologues to G and nothing to T. If the downstream gene of the frameshift does not have homologues and you cannot find a slippery sequence as denoted in the review paper cited in the guide https://seaphagesbioinformatics.helpdocsonline.com/article-54 , do not 'force' a frameshift. I would not call 4Gs, not followed by 3 of something else as a slippery sequence (though we have done it in the past). Is that the case here? debbie

Link to this post | posted 03 Mar, 2020 17:50

debbie

Hi all,
I am going to chime in here, but my understanding today is that every phage may not have the G/T programmed frameshift. In lots of cases we can only find homologues to G and nothing to T. If the downstream gene of the frameshift does not have homologues and you cannot find a slippery sequence as denoted in the review paper cited in the guide https://seaphagesbioinformatics.helpdocsonline.com/article-54 , do not 'force' a frameshift. I would not call 4Gs, not followed by 3 of something else as a slippery sequence (though we have done it in the past). Is that the case here?
debbie

Link to this post \| posted 03 Mar, 2020 18:00
erin.windsor	Ours was identified as CGGGGAG during QC.

Link to this post \| posted 03 Mar, 2020 20:43
stpage	Yeah, that location in Mariokart is just AGGGCCG…. Yago84 did not call the frameshift but there is a aagggcct So maybe I should just follow that.

Link to this post \| posted 03 Mar, 2020 23:40
welkin	I think with those sequences I would go with no slip for now.

Link to this post \| posted 08 Feb, 2022 02:05
Pollenz	Its been ~2 years since this thread and there has been several different scenarios regarding the tail assembly chaperone (TAC) in DR phages. There are currently 8 annotated phages with 5 different TAC annotations (see enclosed file). All of the DR phages have high nucleotide similarity and the same set of genes upstream of the tape measure. All of the genes are from huge PHAMS with 350-550 members across multiple clusters. The 3rd gene upstream of the tape measure has a majority call of hypothetical, only 3 DR phages call this TAC out of 406 members. The 2nd gene upstream has a MAJORITY call of TAC and the gene directly upstream of the tape measure has 55 of 384 calls to TAC. The issue is that in the DR phages, there are FIVE different annotation scenarios for the 8 annotated genomes in this region: 1) No TAC calls to any of the genes 2) TAC call to only the 2nd and 3rd upstream genes (Mariokart) 3) TAC calls with programmed frameshift between 2nd and 3rd upstream genes (Sour and NHagos) 4) TAC call to only the 2nd upstream gene (AnClar) 5) TAC with programmed frameshift between 1st and 2nd upstream genes (CloverMinnie) We are annotating CaiB. We cant have a programmed frameshift between the 2nd and 3rd upstream gens due to an in frame stop codon to the 2nd upstream gene that limits the sequence area that can be used for the slippage to ~25bp (and there is no slippery sequence within this region) We CAN find a non-canonical CGGGGCCG sequence in the 2nd upstream gene that would allow slippage to the 1st upstream gene (just as annotated in CloverMinnie with the SAME slippery sequence. Thoughts, ideas on this DR cluster? There should be consistency based on genes and sequences. Based on lack of wet lab data regarding the slippery sequences in Gordonia, seems that a non-canonical slippery call is preamature. Based on synteny and HHpred, seems that TAC should be called for 2nd upstream gene and possibly 1st upstream, but NOT for 3rd upstream. RS Pollenz 430Kb

Link to this post | posted 08 Feb, 2022 02:05

Pollenz

Its been ~2 years since this thread and there has been several different scenarios regarding the tail assembly chaperone (TAC) in DR phages. There are currently 8 annotated phages with 5 different TAC annotations (see enclosed file). All of the DR phages have high nucleotide similarity and the same set of genes upstream of the tape measure. All of the genes are from huge PHAMS with 350-550 members across multiple clusters. The 3rd gene upstream of the tape measure has a majority call of hypothetical, only 3 DR phages call this TAC out of 406 members. The 2nd gene upstream has a MAJORITY call of TAC and the gene directly upstream of the tape measure has 55 of 384 calls to TAC.

The issue is that in the DR phages, there are FIVE different annotation scenarios for the 8 annotated genomes in this region:

1) No TAC calls to any of the genes
2) TAC call to only the 2nd and 3rd upstream genes (Mariokart)
3) TAC calls with programmed frameshift between 2nd and 3rd upstream genes (Sour and NHagos)
4) TAC call to only the 2nd upstream gene (AnClar)
5) TAC with programmed frameshift between 1st and 2nd upstream genes (CloverMinnie)

We are annotating CaiB. We cant have a programmed frameshift between the 2nd and 3rd upstream gens due to an in frame stop codon to the 2nd upstream gene that limits the sequence area that can be used for the slippage to ~25bp (and there is no slippery sequence within this region)

We CAN find a non-canonical CGGGGCCG sequence in the 2nd upstream gene that would allow slippage to the 1st upstream gene (just as annotated in CloverMinnie with the SAME slippery sequence.

Thoughts, ideas on this DR cluster? There should be consistency based on genes and sequences. Based on lack of wet lab data regarding the slippery sequences in Gordonia, seems that a non-canonical slippery call is preamature. Based on synteny and HHpred, seems that TAC should be called for 2nd upstream gene and possibly 1st upstream, but NOT for 3rd upstream.

RS Pollenz

Link to this post \| posted 10 Feb, 2022 03:30
debbie	Rick, When I look at the data I see gene 25 (19067 - 19582) has a pFam hit to TAC. I see that gene 24 (18604 - 19014) has 3 pFam hits - one to a capsid, one to a tail, and one to HK97gp10 (that we don't know where it is in the structure - a head or tail?) So that is just confusing. I am sure that we overcalled the slippage in CloverMinnie (because at that moment in time, we thought there had to be a slippage (and now we don't)). So I would not use that one for reference. Likewise I think gene 23 (18266 - 18607)is a Hypothetical Protein. debbie

Link to this post | posted 10 Feb, 2022 03:30

debbie

Rick,
When I look at the data I see gene 25 (19067 - 19582) has a pFam hit to TAC.
I see that gene 24 (18604 - 19014) has 3 pFam hits - one to a capsid, one to a tail, and one to HK97gp10 (that we don't know where it is in the structure - a head or tail?)
So that is just confusing.
I am sure that we overcalled the slippage in CloverMinnie (because at that moment in time, we thought there had to be a slippage (and now we don't)). So I would not use that one for reference.
Likewise I think gene 23 (18266 - 18607)is a Hypothetical Protein.
debbie

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