SEA-PHAGES | All posts created by DanRussell

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Link to this post \| posted 06 Apr, 2016 20:16
DanRussell	Hi Lee, Looks like that error is my fault! When I updated the consed on the VM, apparently I didn't copy all files from the old consed directory to the appropriate places. This would only affect uses of phred and phrap, which are rarely used, but would definitely be used when adding reads. To fix this on your VM, run the following commands when logged in as seafaculty, entering the password as necessary: `sudo cp /usr/local/genome_OLD/bin/phred /usr/local/genome/bin/` `sudo cp /usr/local/genome_OLD/bin/phrap /usr/local/genome/bin/` `sudo cp /usr/local/genome_OLD/lib/phredpar.dat /usr/local/genome/lib/` This should put the files in the appropriate places and make it work again. You're the first to discover this probably because 1) we barely do any Sanger reads any more and 2) I usually run consed on my Mac rather than within the VM. Sorry, and good catch, –Dan

Link to this post | posted 06 Apr, 2016 20:16

Hi Lee,

Looks like that error is my fault! When I updated the consed on the VM, apparently I didn't copy all files from the old consed directory to the appropriate places. This would only affect uses of phred and phrap, which are rarely used, but would definitely be used when adding reads. To fix this on your VM, run the following commands when logged in as seafaculty, entering the password as necessary:

sudo cp /usr/local/genome_OLD/bin/phred /usr/local/genome/bin/

sudo cp /usr/local/genome_OLD/bin/phrap /usr/local/genome/bin/

sudo cp /usr/local/genome_OLD/lib/phredpar.dat /usr/local/genome/lib/

This should put the files in the appropriate places and make it work again. You're the first to discover this probably because 1) we barely do any Sanger reads any more and 2) I usually run consed on my Mac rather than within the VM.

Sorry, and good catch,
–Dan

Posted in: Consed → Adding Sanger Reads

Link to this post \| posted 04 Apr, 2016 18:41
DanRussell	Hi Greg, Looks like in that paper we mention using ClustalW which has been retired in favor of ClustalOmega to generate the numbers, then NJPlot to draw the trees. –Dan

Posted in: Papers → Pham and Phylogenetic analysis software

Link to this post \| posted 23 Mar, 2016 18:16
DanRussell	ball.1766 I have a student who has Linux on his home computer and would like to download Phamerator. Could you please send me the appropriate file/password that he would need to be able to get it up and running? Thanks, Sarah Hey Sarah, If he's just installing Phamerator on his own Linux system, then he probably just needs this quick guide rather than any pre-made image/file or anything. It's not too tough to get it up and running on Linux. –Dan

Link to this post | posted 23 Mar, 2016 18:16

DanRussell

ball.1766
I have a student who has Linux on his home computer and would like to download Phamerator. Could you please send me the appropriate file/password that he would need to be able to get it up and running?

Thanks,
Sarah

Hey Sarah,

If he's just installing Phamerator on his own Linux system, then he probably just needs this quick guide rather than any pre-made image/file or anything. It's not too tough to get it up and running on Linux.

–Dan

Posted in: SEA-PHAGES Virtual Machine → Student download of 2016 VM

Link to this post \| posted 07 Mar, 2016 15:25
DanRussell	Hi Greg, If you really want a quick way to check your phages against some others, you could set up a new Phamerator database using the public version of PhamDB we've set up. There's some more info here. I should mention that I would keep all PhamDB databases relatively small for now, fewer than 100 genomes for sure. –Dan Edited 07 Mar, 2016 15:26

Posted in: Phamerator → Analyze our near finished sequence in Phamerator???

Link to this post \| posted 07 Mar, 2016 15:23
DanRussell	Hi all, Ever wanted to just make your own Phamerator database, but then looked at all the necessary code and immediately given up? It's not an easy process, for sure, but a student named James Lamine from Calvin College—under the supervision of Randy DeJong—has made it much easier. Some of you may remember Randy's talk at last year's Symposium about PhamDB, a web-based way to make your own Phamerator databases. It takes GenBank (.gb) files as input, and outputs links that you can point your copy of Phamerator to in order to use the new database you've created. PhamDB has now been published in Bioinformatics. The installation instructions to get it set up and running on your machine are not bad at all. But we've also set up a (sort of) public version of PhamDB if you want to give it a try. Because it doesn't have account-specific permissions yet, we didn't want to make it fully public, lest random people delete your new databases. So if you're interested in using it, just email me (dar78@pitt.edu) and I'll send you the relevant info. Here's a screenshot: –Dan

Link to this post | posted 07 Mar, 2016 15:23

DanRussell

Hi all,

Ever wanted to just make your own Phamerator database, but then looked at all the necessary code and immediately given up?

It's not an easy process, for sure, but a student named James Lamine from Calvin College—under the supervision of Randy DeJong—has made it much easier. Some of you may remember Randy's talk at last year's Symposium about PhamDB, a web-based way to make your own Phamerator databases. It takes GenBank (.gb) files as input, and outputs links that you can point your copy of Phamerator to in order to use the new database you've created. PhamDB has now been published in Bioinformatics.

The installation instructions to get it set up and running on your machine are not bad at all. But we've also set up a (sort of) public version of PhamDB if you want to give it a try. Because it doesn't have account-specific permissions yet, we didn't want to make it fully public, lest random people delete your new databases. So if you're interested in using it, just email me (dar78@pitt.edu) and I'll send you the relevant info. Here's a screenshot:

–Dan

Posted in: Phamerator → PhamDB: Make your own Phamerator databases

Link to this post \| posted 07 Mar, 2016 15:09
DanRussell	Hi Stephanie, Sure! First let me say that it’s always possible that any two auto-annotations on the same exact sequence won’t match 100%. This is because of the way the algorithms work and databases change, so if you had 25 students each run an auto-annotation they might not all match. But…the more likely explanation in your case is that it appears that Watson potentially has 3 tRNAs right after gene 8. Since Phamerator doesn’t show tRNAs, you just see a small gap there in Phamerator, though the Phamerator numbering does include those, so it looks like it skips from gene 8 to gene 12. DNA Master doesn’t number the tRNAs by default, so the first gene after them ends up being #9 (which was #12 in Phamerator). You can kind of see the Phamerator and DNA Master maps lined up below. Because of this, we never really work by gene number. We instead use start/stop coordinates to identify genes, because those are unambiguous. And if you add or delete a gene, the gene numbers will eventually change anyway to accommodate the new/deleted genes, so they’re ephemeral until the very final submission. Hope that helps! —Dan

Link to this post | posted 07 Mar, 2016 15:09

DanRussell

Hi Stephanie,

Sure! First let me say that it’s always possible that any two auto-annotations on the same exact sequence won’t match 100%. This is because of the way the algorithms work and databases change, so if you had 25 students each run an auto-annotation they might not all match.

But…the more likely explanation in your case is that it appears that Watson potentially has 3 tRNAs right after gene 8. Since Phamerator doesn’t show tRNAs, you just see a small gap there in Phamerator, though the Phamerator numbering does include those, so it looks like it skips from gene 8 to gene 12. DNA Master doesn’t number the tRNAs by default, so the first gene after them ends up being #9 (which was #12 in Phamerator). You can kind of see the Phamerator and DNA Master maps lined up below.

Because of this, we never really work by gene number. We instead use start/stop coordinates to identify genes, because those are unambiguous. And if you add or delete a gene, the gene numbers will eventually change anyway to accommodate the new/deleted genes, so they’re ephemeral until the very final submission.

Hope that helps!
—Dan

Posted in: Phamerator → Phamerator and DNA Master gene numbers don’t match

Link to this post \| posted 03 Mar, 2016 18:47
DanRussell	Hey Joe, What 1:1 means is that the first amino acid of the query sequence matches the first amino acid of the subject sequence. This implies that the start site of your query sequence matches the start site of the published subject sequence. Or, like you said, a 1:4 alignment would mean that the first amino acid of the query matches the fourth amino acid of the subject. This means there are three more amino acids in the published subject sequence that are not in your query. This can indicate that you're choosing a different start than the published one. I made a picture that hopefully helps show these different situations. Remember that the grayed-out bases in this picture won't appear in BLAST results, because they're not part of the alignment. But I included them in this picture to give a better sense of how the protein sequences are really lining up. –Dan Edited 03 Mar, 2016 18:49

Link to this post | posted 03 Mar, 2016 18:47

DanRussell

Hey Joe,

What 1:1 means is that the first amino acid of the query sequence matches the first amino acid of the subject sequence. This implies that the start site of your query sequence matches the start site of the published subject sequence.

Or, like you said, a 1:4 alignment would mean that the first amino acid of the query matches the fourth amino acid of the subject. This means there are three more amino acids in the published subject sequence that are not in your query. This can indicate that you're choosing a different start than the published one.

I made a picture that hopefully helps show these different situations. Remember that the grayed-out bases in this picture won't appear in BLAST results, because they're not part of the alignment. But I included them in this picture to give a better sense of how the protein sequences are really lining up.

–Dan

Edited 03 Mar, 2016 18:49

Posted in: Choosing Start Sites → Using BLAST to validate start sites

Link to this post \| posted 03 Mar, 2016 16:15
DanRussell	Hi Joe, I think the value of using BLAST when choosing start sites lies in the fact that you're comparing your choice to the final choices made by other (often experienced) people. So it's not biological evidence, but it's sort of consensus evidence. You could argue, of course, that simply because someone else has called it a certain way is no reason to call it that way too, and the skeptic in all of us resists conforming to the norm. But many of the genomes in GenBank have been very carefully QCed and pored over by those who do this work more than anyone else. So I think of it kind of like checking your work with an expert. If you BLAST, and get 1:1 hits with many published things, it's like, "Oh, good, seems like everyone agrees with this start call. That makes me a little more confident in it." If all the BLAST hits are not 1:1, it's like "Hmmm…looks like everyone else called a different start for some possibly good reason. Maybe I should double-check and really buttress my argument on this one." Two things to keep in mind when using BLAST results for start sites: Who submitted this genome to GenBank? Was it Debbie/Welkin/Graham? Or was it Jimmy FirstPhage? Give more weight to trusted experts. When was this genome submitted? We have more evidence now for making start site calls than we did in the past, so give more weight to newer genomes than older ones. Hope that helps, –Dan

Link to this post | posted 03 Mar, 2016 16:15

DanRussell

Hi Joe,

I think the value of using BLAST when choosing start sites lies in the fact that you're comparing your choice to the final choices made by other (often experienced) people. So it's not biological evidence, but it's sort of consensus evidence. You could argue, of course, that simply because someone else has called it a certain way is no reason to call it that way too, and the skeptic in all of us resists conforming to the norm. But many of the genomes in GenBank have been very carefully QCed and pored over by those who do this work more than anyone else.

So I think of it kind of like checking your work with an expert. If you BLAST, and get 1:1 hits with many published things, it's like, "Oh, good, seems like everyone agrees with this start call. That makes me a little more confident in it." If all the BLAST hits are not 1:1, it's like "Hmmm…looks like everyone else called a different start for some possibly good reason. Maybe I should double-check and really buttress my argument on this one."

Two things to keep in mind when using BLAST results for start sites:

Who submitted this genome to GenBank? Was it Debbie/Welkin/Graham? Or was it Jimmy FirstPhage? Give more weight to trusted experts.
When was this genome submitted? We have more evidence now for making start site calls than we did in the past, so give more weight to newer genomes than older ones.

Hope that helps,
–Dan

Posted in: Choosing Start Sites → Using BLAST to validate start sites

Link to this post \| posted 03 Mar, 2016 16:05
DanRussell	saleadon Hi Everyone, In "Exploring Bacteriophage Biology", one suggested bioinformatics experiment is to design cluster-specific DNA primers. The article mentions that there is a list of existing primers on phagesdb.org under “Phorum”. However, I can't find that. Has it been moved or deleted? Thanks, Steve Hi Steve, That document needs to be updated! Yes, the PhagesDB Phorum has indeed been deleted. And I'm not sure that we'd necessarily recommend that particular experiment either, since it has proved of limited use. It might be an interesting teaching/thought experiment…like: can you locate regions that are conserved in all the members of a subcluster? A cluster? Or not? (i.e., Is it even possible to design cluster-specific primers?) In any event, I know that Alex Peister at Morehouse College has been the de facto go-to person for cluster primers, so she would be a good resource if you're interested in the current state of those attempts. –Dan

Link to this post | posted 03 Mar, 2016 16:05

DanRussell

saleadon
Hi Everyone,
In "Exploring Bacteriophage Biology", one suggested bioinformatics experiment is to design cluster-specific DNA primers. The article mentions that there is a list of existing primers on phagesdb.org under “Phorum”. However, I can't find that. Has it been moved or deleted?
Thanks,
Steve

Hi Steve,

That document needs to be updated! Yes, the PhagesDB Phorum has indeed been deleted. And I'm not sure that we'd necessarily recommend that particular experiment either, since it has proved of limited use. It might be an interesting teaching/thought experiment…like: can you locate regions that are conserved in all the members of a subcluster? A cluster? Or not? (i.e., Is it even possible to design cluster-specific primers?)

In any event, I know that Alex Peister at Morehouse College has been the de facto go-to person for cluster primers, so she would be a good resource if you're interested in the current state of those attempts.

–Dan

Posted in: Phage Biology → PCR Primers

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

Posted in: Annotation → Locating Terminase Gene

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