Link to this post | posted 30 Mar, 2020 02:06

kmaclea

Hi all, We are working on phage SilentRX, which is currently assigned to Unknown (UNK) cluster, with its closest relatives in cluster AP. Students are about to start in earnest on annotation and first-pass gene assignments have been given out, but I have some weirdness I didn't notice before. At first glance it looked like SilentRX had 99 genes. But now it appears not… Autoannotation in DNA Master finds 100 genes with default settings. Phagesdb and Phamerator at first glance appears to find 99 genes, but gene 94 is missing, so this looks like 98 genes. Because of the numbering differences, the gene numbers in DNA master and Phagesdb/Phamerator are not in agreement. I am working out what to do next with gene numbering system. Any suggestions? Other questions: 1. Why is Gene 94 missing? 2. I have the final gene doing weird things. Gene 100 (Auto annotation) appears to be a reverse gene that crosses from position 678 to 65131. Is this likely to be a real gene? The same gene in Phagesdb is called Gene 99. And weirdly enough it has a second gene member in its Pham–SilentRX Gene 98. So it predicts that Genes 98 and 99 (phages db) or presumably 99 and 100 (autoannotation) are members of the same Pham, with one of them crossing position zero. Is this likely to be real? Any thoughts or suggestions appreciated! – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post | posted 30 Mar, 2020 02:57

debbie

Kyle, 1.The 'missing' gene is a tRNA call. As you investigate, you should be able to discern the discrepancy and what to call. It is tricky. It is not uncommon that the numbers between Phamerator and your DNA Master files do not match. In this case you are talking about the right end of the genome, sometimes the discrepancy is at the left end. So you are lucky. Remember that Glimmer and GeneMark make predictions on a 'random sample' of the genome. Because the 'samples' can be different, the auto-annotation can be different. This is important because it is part of the rationale of why we do the work that we do. Remember in the workshop, I asked that you always orient your self to the stop codon, because all other numbers can be different. 2. What are the end types of your genome? Can you figure out your question about the last 2 genomes once you know that? You will want to be very careful how you annotate both ends of your genome. Have you evaluated the ORF at 63636-64280 (reverse direction)? I am not sure that this is/isn't a gene, but I would strongly consider it. Great genes to consider! debbie

Link to this post | posted 30 Mar, 2020 03:33

kmaclea

Thank you, Debbie! Great questions, and we definitely have not investigated yet. I will try to guide the students in thinking about them this week. (Made more "interesting" because we are all doing this remotely, not side-by-side like before!) Side question: We have another Phage that we sequenced at the same time as SilentRX in our DOGEMs pool that was complete. We have recently identified this sequence I've been calling Brother-of-SilentRX as Phage Gole. Given you guys are closed down right now, can we get our DOGEMs Contig 1 assigned as Gole, and then Phamerated at some point? I will also be doing a summer course on phage annotation and could do Gole at that point instead if that makes more sense time-wise based on all you guys are trying to handle at the moment. Thank you, sincerely! Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post | posted 30 Mar, 2020 12:07
debbie	Kyle, Yes! Have you made a phage page for Gole? That is the first step. Second step is to email Dan with the request to post the sequence there. Cool! debbie

Link to this post | posted 31 Mar, 2020 02:43
kmaclea	Dan got in touch, thank you for that! He already worked his magic! Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post | posted 04 Apr, 2020 20:31

kmaclea

debbie 2. What are the end types of your genome? Can you figure out your question about the last 2 genomes once you know that? You will want to be very careful how you annotate both ends of your genome. I missed a little discussion at the workshop about End types. Can you point to some good reading on this? Thank you! Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129

Link to this post | posted 05 Apr, 2020 15:39
DanRussell	Hi Kyle, Here are a couple options: Sherwood Casjens' paper about using terminases to determine end types, but it also sort of explains what the ends types might be: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082370/ A more recent paper about the program PhageTerm, which tries to predict end type from sequencing reads: https://www.nature.com/articles/s41598-017-07910-5 Take care, –Dan

Link to this post | posted 07 Apr, 2020 02:00
kmaclea	Fantastic! Thank you, Dan! Kyle – Kyle MacLea Associate Professor, University of New Hampshire at Manchester kyle.maclea@unh.edu +1 603-641-4129