Link to this post | posted 24 Feb, 2016 21:12

dmonti

Hi Folks - We are attempting to create a final FASTA file for one of our recently sequenced phage and need to locate the terminase to know where to assign position 1 of the genome. After many BLAST searches and a few HHPred searches of nearly every protein with a typical terminase size, I am coming up empty-handed. Are there any other tricks or tips that you can share regarding locating the terminase? We have also tried running a dotplot comparison with other Acinetobacter phage and nothing seems to match. Many thanks for any assistance you can offer! Best, Denise

Link to this post | posted 24 Feb, 2016 22:26
lhughes	Hi Denise, I'm not a lot of help on this, but one thought that came to mind was locating the tapemeasure gene first and then working backward from there to the likely area for the terminase. Lee

Link to this post | posted 25 Feb, 2016 05:35

dmonti

Hi Lee - Thanks for the response. We are even struggling to find a tapemeasure gene but the good news is we finally did locate a terminase gene today! Unfortunately though, some questions remain. I know Welkin was using the terminase gene to determine a start position for a circularly permutated genome. The position of our terminase is at the end of a long string of forward genes with a few reverse genes sprinkled here and there. I am struggling to determine where I should assign position 1 in the genome. Should I 'ignore' the reverse genes for now and assign position 1 at the beginning of the forward genes, or take a different approach and assign position 1 at the short string of forward genes that immediately follows the first tiny reverse gene. Any advice is greatly appreciated! Denise

Link to this post | posted 25 Feb, 2016 14:35
lhughes	I think you are going to need Dan's help to figure this one out. Lee

Link to this post | posted 25 Feb, 2016 16:03

cdshaffer

I had the same issue with some of our recently isolated Streptomyces phage. Several of them were so novel across the whole genome based on BLASTn that I had to go the "find the terminase" route. Looking at some of the previous Streptomyces phage the terminase is not always the very left most gene, so I matched those phage and I ended up moving to the left several genes to find a "clearing" in the coding potential and then pick base 1 somewhere that would avoid dividing any long ORFs. You can look at phage Yara_draft in phamerator as an example. A few of our phage were similar to Yara so we used that as a guide. In Yara the similarity to terminase starts at around 2.5 kb and is the 8th gene called by DNA Master auto-annotation.

Link to this post | posted 25 Feb, 2016 16:03
DanRussell	Hi Denise, Does this happen to be Beth or C3PO? –Dan

Link to this post | posted 25 Feb, 2016 18:19

dmonti

Hi Dan - This is a phage called Lamar that is an Acinetobacter phage. Both Lamar and Beth are very interesting in that they form very distinct halos which we have not seen described for Acinetobacter phage. But the sequences for Beth and Lamar are very distinct; Lamar is about 30,000bp larger than Beth and no sequence similarity that we can see via a quick dot plot. I can send a dnam5 file if you wouldn't mind putting some expert eyes on the sequence. The terminase is gp121, there seems to be a chunk of structural genes around gp117-119 and again around gp88-92. I am just unsure where the 'logical' breakpoint would be. I may take Chris' suggestion and look in some of the other Acinetobacter baumannii phage to see if there is precedence for where to start the genome relative to the terminase. Other suggestions? Denise

Link to this post | posted 25 Feb, 2016 18:34

DanRussell

Hi Denise, First of all, are you sure this is a circularly permuted genome? It's always possible that the phage already has ends of its own, then you don't have to worry about choosing them yourself. I can check that if you give me the sequence file and the sequencing reads. (Posting on Dropbox or Google Drive is a good way to move those large files.) If it truly is circularly permuted, I'm sure Debbie would be happy to take a look at potential Base 1 options. She's done this a lot! –Dan

Link to this post | posted 02 Mar, 2016 21:47

dmonti

HI Dan - My apologies for the delay. After your post, I wanted to go back and add in more reads to see if I perhaps missed the drop in coverage indicating a defined end. It took me a few days to get around to working in the sequence again. I did add in the reads and I see one spike in coverage, but it doesn't look like a terminal repeat. I would greatly appreciate a 'keener' pair of eyes. I will post the original Lamar.fastq file as well as the newly downsampled Lamar_100k.fastq file. Is there another file you need? I have a dnam5 file created from the Lamar_100k contig that I will also upload. Gene 121 is the terminase. Many thanks! Let me know if you want me to send anything else! Denise

Link to this post | posted 02 Mar, 2016 21:49
DanRussell	Hi Denise, Sounds like that would be enough for me to take a look! Let me know when they're posted and I'll see what I can see. –Dan