SEA-PHAGES | All posts created by stpage

Link to this post \| posted 29 Dec, 2023 14:00
stpage	I am trying to get my fastq files into the VM. They seem to be too large to email. I think I followed these instructions for shared folders and I did choose "auto-mount" can see the shared folders but they did not appear in the /Media folder. I updated the virtual machine for Newbler but the shared folders are not under media or desktop or home. I tried searching the whole computer with no success. They are visible in the VM screen in a bottom icon for shared folders (which doesn't show in the screenshot) but…. I tried a couple of online help forums. Seems to be a mounting problem maybe? So I tried following the directions at: https://vineetcic.medium.com/how-to-access-folders-on-ubuntu-host-machine-from-an-ubuntu-virtual-machine-in-virtualbox-a020a9dbc5a4 but I am not at all sure that I did it correctly in command line. Thanks, Shallee —————————————————– cdshaffer easy way (that I use in class when time is an issue) I tell student: open browser, connect to email, send files to yourself as attachment. Bonus feature: automatic backup of files. If you need to move many files back and forth: 1. make sure you are using a recent version of Virtualbox and have installed guest additions in the ubuntu machine 2. with machine off go to settings -> shared folder 3. click the tiny folder with the plus sign to add a shared folder 4. in the folder path entry click the arrowhead and select "other" 5. select the mac Desktop folder to share between guest and host 6. select the "Make permanent" setting so you only have to do this once. 7. The shared folder will be in the /media folder in the guest which is in the top level of folders (i.e. two folders up from your home folder). The name of the folder inside the guest machine will be something like sf_Desktop 8. Files placed in there will appear on the Desktop of your mac You an also set up drag'n'drop to supposedly be able to move files back and forth but I have had less success with that technique EDIT 1/11/24: Ok, didn't get drag'n'drop to work but got the files in with the SharedFolder bit. I suspect my filepaths were wrong…. Edited 11 Jan, 2024 18:27

Link to this post | posted 29 Dec, 2023 14:00

I am trying to get my fastq files into the VM. They seem to be too large to email.

I think I followed these instructions for shared folders and I did choose "auto-mount" can see the shared folders but they did not appear in the /Media folder. I updated the virtual machine for Newbler but the shared folders are not under media or desktop or home. I tried searching the whole computer with no success. They are visible in the VM screen in a bottom icon for shared folders (which doesn't show in the screenshot) but….
I tried a couple of online help forums. Seems to be a mounting problem maybe? So I tried following the directions at: https://vineetcic.medium.com/how-to-access-folders-on-ubuntu-host-machine-from-an-ubuntu-virtual-machine-in-virtualbox-a020a9dbc5a4 but I am not at all sure that I did it correctly in command line.

Thanks,
Shallee

—————————————————–

cdshaffer
easy way (that I use in class when time is an issue) I tell student: open browser, connect to email, send files to yourself as attachment. Bonus feature: automatic backup of files.

If you need to move many files back and forth:
1. make sure you are using a recent version of Virtualbox and have installed guest additions in the ubuntu machine
2. with machine off go to settings -> shared folder
3. click the tiny folder with the plus sign to add a shared folder
4. in the folder path entry click the arrowhead and select "other"
5. select the mac Desktop folder to share between guest and host
6. select the "Make permanent" setting so you only have to do this once.
7. The shared folder will be in the /media folder in the guest which is in the top level of folders (i.e. two folders up from your home folder). The name of the folder inside the guest machine will be something like sf_Desktop
8. Files placed in there will appear on the Desktop of your mac

You an also set up drag'n'drop to supposedly be able to move files back and forth but I have had less success with that technique

EDIT 1/11/24: Ok, didn't get drag'n'drop to work but got the files in with the SharedFolder bit. I suspect my filepaths were wrong….

Edited 11 Jan, 2024 18:27

Posted in: SEA-PHAGES Virtual Machine → Shared folders

Link to this post \| posted 30 Mar, 2023 16:11
stpage	Official function list (2023) "we are no longer using "capsid morphogenesis protein""

Posted in: Functional Annotation → Capsid morphogenesis protein

Link to this post \| posted 30 Mar, 2023 12:46
stpage	Looks like these have been fixed @ NCBI but not in SEA-PHAGES…? 9Kb

Posted in: Cluster DC Annotation Tips → capsid maturation proteases

Link to this post \| posted 30 Nov, 2022 19:47
stpage	Vic wrote: "Like preparing webbed plates, it is important that not all the host bacteria is lysed early. It is therefore important that you setup the experiment early in the day and check on the culture before you leave in the evening. If the culture is clear (and the control is cloudy), then you've run out of host bacteria for phage amplification." Hi, what is the concern with "early lysis"? Is it just that it limits phage amplification or that too many bacterial degradative products are released early or…..? (just wondering, we've had trouble getting students to be consistent with timing) Edited 30 Nov, 2022 19:48

Link to this post | posted 30 Nov, 2022 19:47

stpage

Vic wrote: "Like preparing webbed plates, it is important that not all the host bacteria is lysed early. It is therefore important that you setup the experiment early in the day and check on the culture before you leave in the evening. If the culture is clear (and the control is cloudy), then you've run out of host bacteria for phage amplification."

Hi, what is the concern with "early lysis"? Is it just that it limits phage amplification or that too many bacterial degradative products are released early or…..? (just wondering, we've had trouble getting students to be consistent with timing)

Edited 30 Nov, 2022 19:48

Posted in: Phage Discovery/Isolation → Protocol to grow phage in culture?

Link to this post \| posted 08 Aug, 2022 20:56
stpage	Finally, a lot of published sequences are actually sequences of prophage and base 1 and orientation are set by the location of the insertion site and the standard orientation for the host genome. [based on your gene matching I think this is the case for NC_015296.1] Ah, ok, that is interesting. I hadn't suspected that. I would recommend you set your base 1 and strand using a similar stratagy, that is to say, pick the base 1 and orientation to make the subsequent steps of comparison as easy as possible. But that of course depends on what you're comparing your genome to. The good news here is that DNA master has a nice feature if you want to "roll" the genome around to set a different base 1 as well as the ability to switch to the complementary strand. Great information, Chris. I'll give that a try. Maybe it will also help us pull out a few more functions for structural genes by synteny. Thank you so much for your help.

Link to this post | posted 08 Aug, 2022 20:56

stpage

Finally, a lot of published sequences are actually sequences of prophage and base 1 and orientation are set by the location of the insertion site and the standard orientation for the host genome. [based on your gene matching I think this is the case for NC_015296.1]
Ah, ok, that is interesting. I hadn't suspected that.

I would recommend you set your base 1 and strand using a similar stratagy, that is to say, pick the base 1 and orientation to make the subsequent steps of comparison as easy as possible. But that of course depends on what you're comparing your genome to. The good news here is that DNA master has a nice feature if you want to "roll" the genome around to set a different base 1 as well as the ability to switch to the complementary strand.
Great information, Chris. I'll give that a try. Maybe it will also help us pull out a few more functions for structural genes by synteny. Thank you so much for your help.

Posted in: Newbler → Getting Started with Phage Assembly

Link to this post \| posted 07 Aug, 2022 18:04
stpage	More info added in bold Hi, I have a (circular) genome that was assembled at NCSU and its closest hit is a phage that was annotated in 2010 at Wellcome Trust Sanger back in 2010 (NC_015296.1). So, no UPitt assembly QC on either, I'm afraid. Hopefully, someone will help me wrap my head around this. The strand is completely opposite between the two and there is a relative inversion in the middle. That is, all my forward genes are listed as complement in the other phage and vice versa. But, (with gp210 approximately the ends of both) the gp numbers are partially inverted gp1+strand=gp64-strand, gp2+strand=63-strand….. gp61-=gp1+; gp 62-=gp210+, gp 63=209….etc). So, it is like there are two giant crossovers if you map them in a phamerator fashion. I know that BLASTN searches also the reverse complement, but all of these genes are listed as Q1:S1 essentially, not reverse. So, I think even if I assume that my genome should have gp 61 as gp1, there is still an inversion relative to each other. Otherwise, I would have gp1=gp210, gp2=gp209…. Finally, there are other phages (published more recently but less sequence conservation) that have the same strand but whose halves are flipped relative to mine. gp1+=gp142+, gp27-=gp168-, gp32-=gp172- So, my current hypothesis is that phage A may be in GenBank as reverse complement but that doesn't matter. Coding is strand agnostic, so I think if I had gp1=gp210, gp2=gp209…. But, we have about 80% NKF and so large-scale functional synteny is a challenge but tapemeasure protein appears to be after the transition from forward to reverse around our gp 30. So it is early in the genome with our gp1 but not in the forward direction, and a couple of tail fiber proteins are "after" tapemeasure as reverse genes. So that would indicate that our assembly is maybe entirely reverse complement compared to the consensus setup? Q1: Is it possible that my assembly should be inverted in some way? "Complementing the contig" for the whole genome? (Even if I were to base it on the reverse complement, it would still have an inversion compared to the other.) Q2: Or, should I start with gp1 in the middle and flip the halves? Thanks.

Link to this post | posted 07 Aug, 2022 18:04

stpage

More info added in bold

Hi, I have a (circular) genome that was assembled at NCSU and its closest hit is a phage that was annotated in 2010 at Wellcome Trust Sanger back in 2010 (NC_015296.1). So, no UPitt assembly QC on either, I'm afraid. Hopefully, someone will help me wrap my head around this.

The strand is completely opposite between the two and there is a relative inversion in the middle. That is, all my forward genes are listed as complement in the other phage and vice versa. But, (with gp210 approximately the ends of both) the gp numbers are partially inverted gp1+strand=gp64-strand, gp2+strand=63-strand….. gp61-=gp1+; gp 62-=gp210+, gp 63=209….etc). So, it is like there are two giant crossovers if you map them in a phamerator fashion. I know that BLASTN searches also the reverse complement, but all of these genes are listed as Q1:S1 essentially, not reverse.

So, I think even if I assume that my genome should have gp 61 as gp1, there is still an inversion relative to each other. Otherwise, I would have gp1=gp210, gp2=gp209….

Finally, there are other phages (published more recently but less sequence conservation) that have the same strand but whose halves are flipped relative to mine. gp1+=gp142+, gp27-=gp168-, gp32-=gp172-

So, my current hypothesis is that phage A may be in GenBank as reverse complement but that doesn't matter. Coding is strand agnostic, so I think if I had gp1=gp210, gp2=gp209…. But, we have about 80% NKF and so large-scale functional synteny is a challenge but tapemeasure protein appears to be after the transition from forward to reverse around our gp 30. So it is early in the genome with our gp1 but not in the forward direction, and a couple of tail fiber proteins are "after" tapemeasure as reverse genes. So that would indicate that our assembly is maybe entirely reverse complement compared to the consensus setup?

Q1: Is it possible that my assembly should be inverted in some way? "Complementing the contig" for the whole genome? (Even if I were to base it on the reverse complement, it would still have an inversion compared to the other.)

Q2: Or, should I start with gp1 in the middle and flip the halves?

Thanks.

Posted in: Newbler → Getting Started with Phage Assembly

Link to this post \| posted 07 Aug, 2022 17:41
stpage	Hi, I have a (circular) genome that was assembled at NCSU and its closest hit is a phage that was annotated in 2010 at Wellcome Trust Sanger back in 2010 (NC_015296.1). So, no UPitt assembly QC on either, I'm afraid. Hopefully, someone will help me wrap my head around this. The strand is completely opposite between the two and there is a relative inversion in the middle. That is, all my forward genes are listed as complement in the other phage and vice versa. But, (with gp210 approximately the ends of both) the gp numbers are partially inverted gp1+strand=gp64-strand, gp2+strand=63-strand….. gp61-=gp1+; gp 62-=gp210+, gp 63=209….etc). So, it is like there are two giant crossovers if you map them in a phamerator fashion. I know that BLASTN searches also the reverse complement, but all of these genes are listed as Q1:S1 essentially, not reverse. So, I think even if I assume that my genome should have gp 61 as gp1, there is still an inversion relative to each other. Otherwise, I would have gp1=gp210, gp2=gp209…. Finally, there are other phages (published more recently but less sequence conservation) that have the same strand but whose halves are flipped relative to mine. gp1+=gp142+, gp27-=gp168-, gp32-=gp172- So, my current hypothesis is that phage A may be in GenBank as reverse complement but that doesn't matter. Coding is strand agnostic, so I think if I had gp1=gp210, gp2=gp209…. for the whole genome it would not matter, right? Q1: Is it possible that my assembly should be inverted in some way? "Complementing the contig" for the whole genome? Even if I were to base it on the reverse complement, it would still have an inversion compared to the other. Q2: Or, should I start with gp1 in the middle and flip the halves? Q3: Alternatively, am I thinking about this whole thing stupidly and there really is no consensus on which strand or where to start gp1? Thanks.

Link to this post | posted 07 Aug, 2022 17:41

stpage

Hi, I have a (circular) genome that was assembled at NCSU and its closest hit is a phage that was annotated in 2010 at Wellcome Trust Sanger back in 2010 (NC_015296.1). So, no UPitt assembly QC on either, I'm afraid. Hopefully, someone will help me wrap my head around this.

The strand is completely opposite between the two and there is a relative inversion in the middle. That is, all my forward genes are listed as complement in the other phage and vice versa. But, (with gp210 approximately the ends of both) the gp numbers are partially inverted gp1+strand=gp64-strand, gp2+strand=63-strand….. gp61-=gp1+; gp 62-=gp210+, gp 63=209….etc). So, it is like there are two giant crossovers if you map them in a phamerator fashion. I know that BLASTN searches also the reverse complement, but all of these genes are listed as Q1:S1 essentially, not reverse.

So, I think even if I assume that my genome should have gp 61 as gp1, there is still an inversion relative to each other. Otherwise, I would have gp1=gp210, gp2=gp209….

Finally, there are other phages (published more recently but less sequence conservation) that have the same strand but whose halves are flipped relative to mine. gp1+=gp142+, gp27-=gp168-, gp32-=gp172-

So, my current hypothesis is that phage A may be in GenBank as reverse complement but that doesn't matter. Coding is strand agnostic, so I think if I had gp1=gp210, gp2=gp209…. for the whole genome it would not matter, right?

Q1: Is it possible that my assembly should be inverted in some way? "Complementing the contig" for the whole genome? Even if I were to base it on the reverse complement, it would still have an inversion compared to the other.

Q2: Or, should I start with gp1 in the middle and flip the halves?

Q3: Alternatively, am I thinking about this whole thing stupidly and there really is no consensus on which strand or where to start gp1?

Thanks.

Posted in: Newbler → Getting Started with Phage Assembly

Link to this post \| posted 26 May, 2022 14:38
stpage	I never got a response from MPI. I tried it with and without logging in and it didn't seem to matter. Instead of the usual copying over the previous input, I started hitting HHPRED again to reload the search tool and not using the job ID and it seems to be ok now. (it is saving the three databases but I have to keep track of which GP). (It is also possible that it was a transient server or traffic problem on their end). But it's working now! Edited 26 May, 2022 15:05

Link to this post | posted 26 May, 2022 14:38

stpage

I never got a response from MPI.
I tried it with and without logging in and it didn't seem to matter.
Instead of the usual copying over the previous input, I started hitting HHPRED again to reload the search tool and not using the job ID and it seems to be ok now. (it is saving the three databases but I have to keep track of which GP).

(It is also possible that it was a transient server or traffic problem on their end). But it's working now!

Edited 26 May, 2022 15:05

Posted in: Functional Annotation → HHPRED

Link to this post \| posted 19 May, 2022 17:02
stpage	They never emailed me back but we seem to be ok if we are rather gentle with HHPRED. Specifically, don't: -copy over a previous input and resubmit under a new ID -launch multiple HHPRed searches simultaneously under the same browser Sometimes, it seems to help to erase the previous job before entering a new job and launching. It's nice that I can still find inventive ways to break things.

Posted in: Functional Annotation → HHPRED

Link to this post \| posted 11 May, 2022 12:41
stpage	Um, has anyone ever gotten kicked out of HHPRED? My student and I were launching multiple HHPRED jobs (3-4) in multiple tabs and now both of our IPs have been blocked apparently. I can search on a different laptop but when we search on those two laptops, we get "ERROR!". We tried to clear cookies and restart and nope, HHPRED still throws the error, so I think it might be IP-based. I get the error whether I am logged into an HHPRED account or not. If we are the first, um, well, word to the wise. Edited 11 May, 2022 12:43

Link to this post | posted 11 May, 2022 12:41

stpage

Um, has anyone ever gotten kicked out of HHPRED? My student and I were launching multiple HHPRED jobs (3-4) in multiple tabs and now both of our IPs have been blocked apparently. I can search on a different laptop but when we search on those two laptops, we get "ERROR!". We tried to clear cookies and restart and nope, HHPRED still throws the error, so I think it might be IP-based. I get the error whether I am logged into an HHPRED account or not.

If we are the first, um, well, word to the wise.

Edited 11 May, 2022 12:43

Posted in: Functional Annotation → HHPRED

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