Link to this post | posted 03 Jun, 2019 19:25

mdgainey

I am finishing up the new Micro singleton FuzzBuster that has some similarities to the cluster EI phages. The tail assembly chaperone genes are orphams, but do have some blast hits to those found in the EI phages,EC phages, and one singleton Hendrix. None of the EI's or EC's call a frame shift but Hendrix does. I have attached a powerpoint with some screen shots of Fuzzbuster, Hendrix, and an EI. The EI's don't have a glycine in the same area as FuzzBuster and Hendrix. My question is would you call a frameshift here, and if so how would you call it -1? Thanks, Maria 130Kb

Link to this post | posted 26 Feb, 2019 16:18
mdgainey	Thanks Chris, I will wait a few hours and try it again=)

Link to this post | posted 26 Feb, 2019 15:19
mdgainey	I am having some trouble with the starterator reports in PECAAN, the phagesdb gene list, and on the WUSTL site. I checked on phamerator and the phams#'s I am using are still correct. Is anyone else having this issue? I was hoping to check my student's gene calls today.

Link to this post | posted 02 Jan, 2019 20:53

mdgainey

Hi all, I am currently working on QCing cluster N BabeRuth from our 2018 faculty hackathon. On the official functions list is says the Xeno_32 is the example for the toxin in toxin/antitoxin system, HicA-like, but this is not called in Phamerator. BabeRuth_40 is in the same pham as Xeno_32 (Pham 3607), we have currently annotated this as a membrane protein but could switch it to HicA-like if this is correct. In the prophage-mediated defense cluster N paper in figure 4 I think they are just labeled these as membrane proteins. Is this an error on the official functions list? I think BabeRuth_37 should be assigned the HicA-like toxin/antitoxin function based on HHPRED data and these are labeled as toxin in figure 4 of the paper, but see no such support for 40. BabeRuth_40 MENVPPSPPPGWYPDPVGSGGQRYWDGQRWTEHYAPPAVAAQIADRRFTVNYGFALLAFFSLLATVGLPLLAMAGGAGADVGPFAILWMLWGGMWTLVWTAFAIQHTLRNRR BabeRuth_37 MNRRIESLGGVQTRQRGSHRRYAVTYTDEMGIVRSAFTTVQQHKSQEIPLGTLRAIQRDLEPAFGKGWLLG

Link to this post | posted 12 Oct, 2018 22:22

mdgainey

Hi I thought I would just add a bit to this thread since I had some issues with my preps last year. For our initial digest analysis this year I think I am just going to omit the DNase and RNase treatment altogether, and instead of precipitating the DNA I am going to do the 4 degree concentration spin that the guide suggests, and then prep/digest. I had good luck with this, this summer with some of my Microphages that I had issues with last semester and got nice clean yields. For the phages selected for sequencing, I will use the protocol as Vic has suggested. Just thought I would throw this out there as another option to try.

Link to this post | posted 18 Jul, 2018 19:08

mdgainey

Thus far it appears that none of the EA phages are reported to contain tRNAs. We recently finished annotating an EA2 bacteriophage Andromedas, that had a potential tRNA that was not called by Aragorn, but was called by tRNA scanSE but bc it had an infernal score of 38 and we decided to not include it in our annotation after looking at the structure of the anticodon loop. I am currently checking an EA5 phage, Neferthena, and again we found a potential tRNA that was not called by Aragorn, but was called by tRNAscanSE, this time the infernal score is 52. I have attached screen shots of the two tRNAs from PECAAN, and would like a second opinion on whether or not to keep the tRNA from Neferthena, the anti-codon loop does not look canonical to me, but I don't have much experience looking at these structures. Any advice is appreciated. Thank you, Maria 366Kb

Link to this post | posted 17 Jul, 2018 20:42
mdgainey	Thanks Welkin, Maria

Link to this post | posted 17 Jul, 2018 16:32
mdgainey	Thanks Welkin, Maria

Link to this post | posted 17 Jul, 2018 15:50

mdgainey

I have been annotating a cluster K5 genome (Rando14). According to the functional assignments document, if we can only find 1 terminase, then we should simply label it terminase. However, a lot of the K's have called gene5 pham 40672(shown in attached powerpoint) a small terminase, but the HHPRED does not support this (attached). I took a look at old forum posts from some faculty working on calling cluster J terminases and found a post from Jacqueline Washington that suggested looking at this paper where they found a small terminase subunit that did not have an HTH binding motif, but did have a coiled coil motif " Archives of Virology 2012 Nov. 157 (11) in which they explained that most terminase small subunits have HTH DNA binding motifs but some don't. They used the program COILS2 (http://www.ch.embnet.org/software/COILS_form.html) to identify the small terminase as it reveals the presence of a coiled coil motif (CCM) which is need for protein dimerization." I ran gene 5 through this and it does indeed seem to have a nice CCM motif (output attached). Is this enough evidence to call this a small terminase subunit, or am I just following the leader here? Thank you, Maria 458Kb

Link to this post | posted 13 Jul, 2018 18:22

mdgainey

Hi I have been annotating Rando14, a K5 bacteriophage and when we go to the area where many of the K5's have an RtcB ligase, there were some weird reverse genes that we needed to delete, and then we found 2 forward ORFs that we think should fill this area nicely with good CP, the first in the +2, and the second in the +1 reading frame that blast up to the N and C terminal portions of the the RtcB ligase respectively. I have attached a power point showing the potential slippery GGGG sequence, where I think the frame shift might occur, and the protein products. Do you think this could possibly be a sequencing error, or perhaps a real frameshift? I know we don't have wet bench data to support frameshifts, other than in the tail assembly chaperones, so I would probably not annotate this as such, but since this one has a predicted function I thought I would post about it to get some advice on how to handle this one. Thank you, Maria 324Kb