Link to this post | posted 05 May, 2020 01:41

WCUPhage

Hi Debbie and Fred, Yes, the hits I am seeing are for an RNA sigma factor. This protein lies next to several DNA metabolism genes (primpol, helicases, etc.) and reminds me of the T7 system (also a podo). T7 brings in its own RNA polymerase (gene 1) that is used to transcribe its genes, and the major DNA replication genes lie near the RNA pol (gene 2.5=ssDNA binding protein, gene 4=primase/helicase, and gene 5: DNA polymerase). If this protein is a sigma factor, then perhaps this is a similar type of system in these phages. The podos we have discovered do not bring in an RNA polymerase, but a sigma factor that can direct transcription (via the host RNA polymerase?) of its DNA metabolism genes. It seems that this protein, and the DNA metabolism genes I mention above, are unique to the podos in EK, EK1, EK2, and EM. Debbie, I agree with your decision to call this a hypothetical protein based on the current data (or lack thereof). If I had additional solid data, along with the HHPRED, then I'd feel better about assigning function. Jamie

Link to this post | posted 04 May, 2020 14:13

WCUPhage

Hi, I'm finalizing a genome for an EK1 phage, and gene 1 has been historically annotated as NKF. For our phage (Pabst), we have significant HHPRED hits for the gene (see attached screenshot) that suggests this gene may be a sigma factor. There are many hits like this as you scroll through the HHPRED list. The NCBI blast hits tell a similar tale, although the statistics are not as good. With Blast we see E-values of 0.02-0.09 for hits to sigma factors. The conserved domain database does not find anything. I was hoping to hear input on how to treat this gene: leave as NKF or assign a function? I'd feel better about assigning a function if both the HHPRED and Blast results gave a clear answer, and for this reason am leaning toward leaving as NKF. However, I don't want to ignore the HHPRED data and therefore wanted to share with the community to get feedback. Thank you! Jamie Wallen 216Kb

Link to this post | posted 01 May, 2020 20:52
WCUPhage	Thank you, Debbie! I'll call it a tail terminator.

Link to this post | posted 01 May, 2020 13:58

WCUPhage

Hi everyone, I am finishing up a new Cluster EE phage (PoRanda), and I had a question about a functional call. I am looking at gene 7 in PoRanda, and its aa sequence is below. In comparing this gene to other EE's in phamerator, as well as blast searches etc., the majority of the data says I should call this as a tail terminator. However, in the functional assignments sheet it says that to be called a tail terminator it "must have an HHPRED alignment to one of the following: SPP1 17 (5A21 chain G in the macromolecular complex) or Lambda U (3FZ2_chains A through F)". In the HHPred hits for this gene, I do see 3FZ2, with a probability just under 90%. However, the E-values for this hit (and all HHPred hits) are not good. The E-value for the 3FZ2 hit is 8.4. 5A21 is also present, with a coverage of 86.6% and an E-value of 13. Looking at the pham (Pham # 19507), the majority of genomes have this annotated as a tail terminator, while others call it a minor tail protein. I'm looking for guidance on how to call this, given then HHPred results described above. Thank you! MSTLGDLRRELADDVAGSPVELTTYDHVPARVQLPAAFVMAGAPYVEADQTFGSSIVRFGVVLLTQPSMNADETDQLDERIESVQRRLLSAGWLVERIERPEIQDLNGAEVLATALSVAAGGVTFP

Link to this post | posted 05 Feb, 2020 16:17
WCUPhage	Hi, Thank you for the reply, and that was it! They had the correct function typed into PECAAN (based on the official function list), but they had the first letter capitalized. It was not capitalized in the function drop down. It's always something simple. Thank you!

Link to this post | posted 05 Feb, 2020 03:52

WCUPhage

Hi all, We have seen the same issue with the Starterator/Phages DB error in PECAAN, and Chris your answer makes sense. We are seeing another issue in PECAAN where some of the genes will turn to a green check mark, while others will not and stay as a red X. We've looked through carefully and we've added all the information for each gene. As of now about a 1/3 of the genes in the genome will not turn to a green check mark. Any thoughts on this? Jamie Wallen (WCU)

Link to this post | posted 18 Jul, 2018 17:34

WCUPhage

I had a question about functional calls for two genes in phage iPhane7. I have HHPred evidence for a potential function that are not options in the Function list. The genes are: CDS 44394 - 44951 There is evidence of a possible function from HHPRed, which was checked as evidence. The function is "Rep_Org_C ; Putative replisome organiser protein C-terminus". CDS 65032 - 65529 HHPred gives strong functional evidence that this is a DNA processing protein A, but this is not in the list of functions. It also has HHPred evidence for a metal carrier protein, not sure what to do about the functional call. It has been annotated as a ssDNA binding protein in Mr Magoo. Jamie Wallen Edited 18 Jul, 2018 17:34