Link to this post | posted 01 Aug, 2022 12:58

uOttawaPHAGE

Hi Dan. We are finally making our libraries and I was curious if you use the Bioanalyzer to check the library - based on the question above from Fernando it sounds like you do. Some questions: 1. I've not worked with data off the bioanalyzer - is there a cut-off for how you determine if a library is good enough to sequence? 2. Do you use the Qubit before or after library prep? for some reason I thought you used it after, but I'm thinking I have that wrong and you use it before to assess the quality/quantity of the genome. thanks again for your help with these questions. Adam

Link to this post | posted 01 Aug, 2022 12:55

uOttawaPHAGE

Hi Dan. We are finally making our libraries and I was curious if you use the Bioanalyzer to check the library - based on the question above from Fernando it sounds like you do. Some questions: 1. I've not worked with data off the bioanalyzer - is there a cut-off for how you determine if a library is good enough to sequence? 2. Do you use the Qubit before or after library prep? for some reason I thought you used it after, but I'm thinking I have that wrong and you use it before to assess the quality/quantity of the genome. thanks again for your help with these questions. Adam

Link to this post | posted 10 Apr, 2022 13:19
uOttawaPHAGE	yes, 17167. I was referring to the G at the start of the slippery sequence. We will call this frameshift for both Ottawa and Kharcho. The 54457 Pham has 22 members. 2 have called the frameshift (phiSASD1 and Dubu), the rest have not. Should we investigate and make those calls too? Also, any opinion about the very small extension? Is homology to lambda unsual for Actino TACs?

Link to this post | posted 08 Apr, 2022 20:49

uOttawaPHAGE

We think it is GGGAAA at 17164, which would yield this sequence: MSTVTEQFIEGATADSYAADGSKTAEFSPVGAEPIAAETEGYKAPGGGLRESIRDRIAKRRPYASREVFVPAWEETIEVRSISLGLRNEMLTSVMDEETKEADIKKLYPELIIRTSYDPQTGERVFSNDDLAFINGLDAGSADLLAKPALELSGMTDDAKDKEAGKILRDGNLRLAFEVAERTGRTLDELFHGGPSCQPMSSREFTQWRALIEIVEPYEREQAEAKSKTSR The extra C-terminal bit doesn't bring up anything on Blastp (because it has never been called), but on Hhpred there is homology to minor tail protein T, though is this the gpT from lambda? In which case it does match a TAC (I had that wrong above). Edited 08 Apr, 2022 20:50 2.50Mb

Link to this post | posted 08 Apr, 2022 18:18

uOttawaPHAGE

We are annotating Kharcho and Ottawa, which form a new FM cluster. gp40 (Kharcho) and gp44 (Ottawa) are tail assembly chaperones. We have found a slippery sequence and the extension would go up to gp41 and 45, respectively, but the extension has homology to a minor tail protein, not a TAC. Should we call the frameshift? None of the other pham members do (not sure we actually checked them all). We may have some experimental evidence that the frameshift occurs, but not yet conclusive. And weird because in E. coli it looks like the ratio of long:short is ~10-20, the opposite of what has been shown for previously tested TACs.

Link to this post | posted 06 Apr, 2022 18:28
uOttawaPHAGE	Thanks Dan, sort of embarrassing….When I said nearly identical I was basing that not on looking at the aa comparison but on the e values in blastp, and that they are almost the same length. Obviously low e values don't mean identity and I should have looked at this with the students. thanks for your help on this. Still learning…A

Link to this post | posted 06 Apr, 2022 16:36
uOttawaPHAGE	I think the alignment is nearly identical, which is why we think it is an error. Do errors like this happen?

Link to this post | posted 06 Apr, 2022 16:35
uOttawaPHAGE	OK, I understand. He did talk about this in his faculty meeting talk, and Erika ran some of the end programs…I will check with her about she found. So this is common, and they should be identical. For the annotation, I assume we still submit those last two genes. A

Link to this post | posted 06 Apr, 2022 16:19
uOttawaPHAGE	thanks Debbie! I think you meant to link to a page - I assume it is in the Bioinformatics guide, and I should have looked there first…

Link to this post | posted 05 Apr, 2022 21:59

uOttawaPHAGE

We are annotating the two FM cluster phages, Ottawa and Kharcho which both have two duplicated genes at the start (1 and 2) and end (105 and 106) of the phage. They are identical in protein sequence(head-to-tail stopper, minor capsid protein), so even though the h-t stopper can appear only once we will annotate both with the same functions. Is this architecture common? I assume there was a recombination event that might have happened during circularization or excision? Can someone point us to other phages that have the same architecture?