SEA-PHAGES | All posts created by DanRussell

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Link to this post \| posted 09 Feb, 2017 15:32
DanRussell	Note that there's also a little document on using Inkscape with Phamerator maps that came out of the Faculty Retreat last year: http://seaphages.org/faculty/information/#modifications http://seaphages.org/media/docs/UsingInkscapeToEditPhameratorMaps.pdf –Dan

Posted in: Phamerator → Printing phamerator maps

Link to this post \| posted 07 Feb, 2017 16:39
DanRussell	Hi Joe, As for the BLASTing, you could try an intermediate approach between all-genes and one-at-a-time. Welkin made a video about how to BLAST a batch of genes in DNA Master, so you could try chunks of 10 for example. Batch BLASTing in DNA Master As to the E-value, those might just be a result of having a less-common cluster of phage this time around. If you had a new Singleton, you'd expect to see some genes with not-good BLAST hits. Cluster L3s are not that common (only 4 in GenBank, I believe), so it's totally possible that those are "new" genes. Check them out in Phamerator and see if they're orphams. (And an e-value of 1 just means that you'd expect about 1 hit this good against a database this size by random chance…meaning it is indeed a dubious result.) –Dan

Link to this post | posted 07 Feb, 2017 16:39

DanRussell

Hi Joe,

As for the BLASTing, you could try an intermediate approach between all-genes and one-at-a-time. Welkin made a video about how to BLAST a batch of genes in DNA Master, so you could try chunks of 10 for example.

Batch BLASTing in DNA Master

As to the E-value, those might just be a result of having a less-common cluster of phage this time around. If you had a new Singleton, you'd expect to see some genes with not-good BLAST hits. Cluster L3s are not that common (only 4 in GenBank, I believe), so it's totally possible that those are "new" genes. Check them out in Phamerator and see if they're orphams.

(And an e-value of 1 just means that you'd expect about 1 hit this good against a database this size by random chance…meaning it is indeed a dubious result.)

–Dan

Posted in: DNA Master → BLASTing whole genome with "secure" connection

Link to this post \| posted 03 Feb, 2017 17:33
DanRussell	I think Chris is right here, and you unfortunately can't add Cluster info via GenBank file, which is the only input for PhamDB. So you're left with the option of locally modifying your database and adding in the clusters. The problem there is that if you then update the database using PhamDB, and your Phamerator imports the newly-updated version, you'll probably lose those changes and have to add them again. Not ideal! –Dan

Link to this post | posted 03 Feb, 2017 17:33

DanRussell

I think Chris is right here, and you unfortunately can't add Cluster info via GenBank file, which is the only input for PhamDB. So you're left with the option of locally modifying your database and adding in the clusters.

The problem there is that if you then update the database using PhamDB, and your Phamerator imports the newly-updated version, you'll probably lose those changes and have to add them again. Not ideal!

–Dan

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

Link to this post \| posted 27 Jan, 2017 15:24
DanRussell	mavrodi So, just to clarify: if Step 1 fails then the only way to install SEA VM is to start changing BIOS settings? No other options available? I currently have five students in my class that can't install the 64-bit VM. Hi Dmitri, Yes, unfortunately the systems we tested the new VM on didn't have the no-64-bit-option issue, so we didn't anticipate this level of trouble. A second option would be to revert to the 2016 SEA VM, which is fairly similar and won't need much tweaking to be brought up-to-date. If you'd like links to the 2016 version, please email me! –Dan

Link to this post | posted 27 Jan, 2017 15:24

DanRussell

mavrodi
So, just to clarify: if Step 1 fails then the only way to install SEA VM is to start changing BIOS settings? No other options available? I currently have five students in my class that can't install the 64-bit VM.

Hi Dmitri,

Yes, unfortunately the systems we tested the new VM on didn't have the no-64-bit-option issue, so we didn't anticipate this level of trouble. A second option would be to revert to the 2016 SEA VM, which is fairly similar and won't need much tweaking to be brought up-to-date. If you'd like links to the 2016 version, please email me!

–Dan

Posted in: SEA-PHAGES Virtual Machine → No Ubuntu 64-bit option when installing 2017 VM on Windows

Link to this post \| posted 17 Jan, 2017 20:59
DanRussell	Hi Greg, You actually want Hyper-V turned OFF, not on, so I don't think it is an issue that some hardware setups don't allow it. The second step in the instructions above is usually the one that needs to be rectified, and Windows 10 Home users should still be able to do that. –Dan

Posted in: SEA-PHAGES Virtual Machine → No Ubuntu 64-bit option when installing 2017 VM on Windows

Link to this post \| posted 01 Dec, 2016 16:25
DanRussell	Hi all, We switched the SEA Virtual Machine to be 64-bit this year instead of 32-bit, and one unintended side-effect is that on some Windows computers, you'll have to make one or two changes to be able to install the VM. If you'd like this in document version it's at the link below. Instructions to enable 64-bit Ubuntu in Virtual Box Now here's the fix itself… PROBLEM: After installing Virtual Box on Windows, there is no “Ubuntu (64-bit)” option in the dropdown menu, only “Ubuntu (32-bit)”. There are two steps below that should fix this and allow you to select a 64-bit option for your VM installation. The first step can be done from within Windows fairly quickly. If that doesn’t work, the second step required entering your system’s BIOS, so we recommend trying the first one, then moving on to the second only if necessary. Step 1: Change Hyper-V settings (easier fix) In Windows, open the “Turn Windows Features On of Off” window. You can get there by either going to Start Control Panel Programs Turn Windows Features On/Off, or searching for “Windows Features”. Locate the Hyper-V checkboxes and make sure they are all UNCHECKED. See the screenshot below. Restart Virtual Box and see if the 64-bit option is enabled. If yes, yay! If not, go to Step 2. In either case, leave these unchecked. Step 2: Enable Intel virtualization (more complicated fix) Enter your computer’s BIOS (Basic Input/Output System). This may also be called UEFI. The ways to do this vary by manufacturer, but common ways to do this include holding down the Shift key then restarting the computer, or pressing the Fn or F2 key while the computer powers on. It always involves restarting and if you see a message like “Press F2 to enter setup” then do so! If you see Windows, you’re too late, just restart and try again. If you’re having trouble, Google your computer’s make/model and BIOS. For example Google “Enter BIOS on HP Envy 17” and follow the instructions. In the BIOS/UEFI, find the settings regarding Virtualization. These may be in a section called Security. The two settings you are looking for are called: Intel (R) Virtualization Technology Intel (R) VT-d Feature Make sure these are both ENABLED. Exit the BIOS and boot into Windows. Now Virtual Box will have an “Ubuntu (64-bit)” option in the dropdown menu. Edited 01 Dec, 2016 16:40

Link to this post | posted 01 Dec, 2016 16:25

DanRussell

Hi all,

We switched the SEA Virtual Machine to be 64-bit this year instead of 32-bit, and one unintended side-effect is that on some Windows computers, you'll have to make one or two changes to be able to install the VM. If you'd like this in document version it's at the link below.

Instructions to enable 64-bit Ubuntu in Virtual Box

Now here's the fix itself…

PROBLEM: After installing Virtual Box on Windows, there is no “Ubuntu (64-bit)” option in the dropdown menu, only “Ubuntu (32-bit)”.

There are two steps below that should fix this and allow you to select a 64-bit option for your VM installation. The first step can be done from within Windows fairly quickly. If that doesn’t work, the second step required entering your system’s BIOS, so we recommend trying the first one, then moving on to the second only if necessary.

Step 1: Change Hyper-V settings (easier fix)

In Windows, open the “Turn Windows Features On of Off” window.
Locate the Hyper-V checkboxes and make sure they are all UNCHECKED. See the screenshot below.

Restart Virtual Box and see if the 64-bit option is enabled. If yes, yay! If not, go to Step 2. In either case, leave these unchecked.

Step 2: Enable Intel virtualization (more complicated fix)

Enter your computer’s BIOS (Basic Input/Output System).
In the BIOS/UEFI, find the settings regarding Virtualization.

Intel (R) Virtualization Technology
Intel (R) VT-d Feature

ENABLED

Exit the BIOS and boot into Windows. Now Virtual Box will have an “Ubuntu (64-bit)” option in the dropdown menu.

Edited 01 Dec, 2016 16:40

Posted in: SEA-PHAGES Virtual Machine → No Ubuntu 64-bit option when installing 2017 VM on Windows

Link to this post \| posted 17 Nov, 2016 17:00
DanRussell	Nicholas Edgington Dan: With such great sequence coverage that has been seen from past years, would the contaminating host DNA really cause much of a problem with assembly? Maybe one could just add a bit of RNAse directly to the HTL's? It would be interesting to know if you have ever tried phage genomic sequencing with and without the addition of the DNAse/RNAse cocktail, and compared the assembly results. Hey Nick, We haven't tried that in any real experimental way. We do sometimes get samples with host DNA contamination, and in most cases it's relatively low coverage compared to the phage and thus isn't a problem. On the other hand, we have seen a few samples where the host coverage was high enough to drop the phage coverage to an unusable/undetectable level. So I guess: who knows? Also, has anyone tried side-by-side old protocol versus new protocol to check for yields from each? That would perhaps shed some light. Having a high titer is certainly the most important thing to getting good yield, so bumping up the titer wherever possible is probably the best way to help. –Dan

Link to this post | posted 17 Nov, 2016 17:00

DanRussell

Nicholas Edgington
Dan: With such great sequence coverage that has been seen from past years, would the contaminating host DNA really cause much of a problem with assembly? Maybe one could just add a bit of RNAse directly to the HTL's? It would be interesting to know if you have ever tried phage genomic sequencing with and without the addition of the DNAse/RNAse cocktail, and compared the assembly results.

Hey Nick,

We haven't tried that in any real experimental way. We do sometimes get samples with host DNA contamination, and in most cases it's relatively low coverage compared to the phage and thus isn't a problem. On the other hand, we have seen a few samples where the host coverage was high enough to drop the phage coverage to an unusable/undetectable level. So I guess: who knows?

Also, has anyone tried side-by-side old protocol versus new protocol to check for yields from each? That would perhaps shed some light. Having a high titer is certainly the most important thing to getting good yield, so bumping up the titer wherever possible is probably the best way to help.

–Dan

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 16 Nov, 2016 15:12
DanRussell	Hi all, Since we at Pitt are receiving DNA samples for sequencing (and thus gel pictures as well) I can say that we're seeing more smeary degraded stuff this year than in the past. Not sure what's going on, but I don't think it's limited to 1-2 schools. –Dan

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 16 Nov, 2016 15:08
DanRussell	Hi all, If you're experiencing recent problems with DNA Master, it's probably because of the update of NCBI to use secure connections. See the blog post and forum below, but the gist of it is that DNA Master was updated on Nov 15, 2016 to be able to handle this, so if you haven't updated your DNA Master since then, please do so! http://seaphages.org/blog/2016/11/16/dna-master-updated-use-secure-ncbi-connections/ http://seaphages.org/forums/topic/214/ –Dan

Link to this post | posted 16 Nov, 2016 15:08

DanRussell

Hi all,

If you're experiencing recent problems with DNA Master, it's probably because of the update of NCBI to use secure connections. See the blog post and forum below, but the gist of it is that DNA Master was updated on Nov 15, 2016 to be able to handle this, so if you haven't updated your DNA Master since then, please do so!

http://seaphages.org/blog/2016/11/16/dna-master-updated-use-secure-ncbi-connections/

http://seaphages.org/forums/topic/214/

–Dan

Posted in: DNA Master → Glimmer Failure on Auto Annotation

Link to this post \| posted 16 Nov, 2016 15:06
DanRussell	New as of November 15, 2016 Some very important changes have been made to DNA Master to accommodate NCBI's new secure https protocols. Versions of DNA Master from before this date will not be able to connect to NCBI and thus will not be able to auto-annotate genomes or run BLAST. Updating your DNA Master will allow you to be able to use NCBI's new protocols. See this blog post for more information. http://seaphages.org/blog/2016/11/16/dna-master-updated-use-secure-ncbi-connections/ –Dan

Link to this post | posted 16 Nov, 2016 15:06

DanRussell

New as of November 15, 2016

Some very important changes have been made to DNA Master to accommodate NCBI's new secure https protocols. Versions of DNA Master from before this date will not be able to connect to NCBI and thus will not be able to auto-annotate genomes or run BLAST.

Updating your DNA Master will allow you to be able to use NCBI's new protocols. See this blog post for more information.

http://seaphages.org/blog/2016/11/16/dna-master-updated-use-secure-ncbi-connections/

–Dan

Posted in: DNA Master → Important DNA Master Update

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