SEA-PHAGES | All posts created by DanRussell

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Link to this post \| posted 28 Jan, 2022 14:46
DanRussell	Hi Amaya, You can check, but my guess is that your student has an M1 Mac. These are the newer Macs using Apple chips instead of intel, and VirtualBox won't work on those. See this forum post for some info: https://seaphages.org/forums/topic/5256/ –Dan

Posted in: SEA-PHAGES Virtual Machine → Error message when installing VirtualBox in MacBook Air

Link to this post \| posted 27 Jan, 2022 18:46
DanRussell	Hi all, Christina Spencer from LeTourneau has compiled some troubleshooting and/or resources that were useful for her as she was helping students with Macs get DNA Master running. They're below. Kernel Driver Error Trouble-Shooting: https://seaphages.org/forums/topic/5313/?page=1#post-9204 Forum about error with DNA Master regarding FTP Connection (assuming Pitt server is not down at the time): https://seaphages.org/forums/topic/5155/?page=1#post-8693 Forum about errors primarily with DNA Master once downloaded: https://seaphages.org/forums/topic/5109/ How to resize Windows/fix seamless mode: Based off the SeaPhages forum, you need the “auto-resize guest display” to be checked from the view tab at the top of your Mac while in the Windows emulator. If it is checked, but it is greyed out (from the linuxquestions), click the command (host button) and “L” button combination a few times until the screen resizes https://seaphages.org/forums/topic/5109/, [SOLVED] VirtualBox: Seamless Mode grayed out, Host Additions are installed (linuxquestions.org) Forum about using WINE instead of VirtualBox: https://seaphages.org/forums/topic/17/ Also, this forum has some general information: https://seaphages.org/forums/topic/4761/ Forum about M1 Macs “unsupported architecture” https://seaphages.org/forums/topic/5256/. Parallels costs money in a yearly subscription. Other workarounds still being investigated as of Jan. 2022 on this forum Troubleshooting: if you start your DNA Master file and it says, “No permission”, you have the file saved in a spot that you do not have access to. A work-around is every time you start DNA Master, you will have to right-click on the start icon and click “run as administrator.” Also, can follow this forum to set the icon properly: https://seaphages.org/forums/topic/78/?page=1#post-506 Setting DNA Master Preferences: https://seaphagesbioinformatics.helpdocsonline.com/article-66 Thanks, Christina! –Dan

Link to this post | posted 27 Jan, 2022 18:46

DanRussell

Hi all,

Christina Spencer from LeTourneau has compiled some troubleshooting and/or resources that were useful for her as she was helping students with Macs get DNA Master running. They're below.

Kernel Driver Error Trouble-Shooting: https://seaphages.org/forums/topic/5313/?page=1#post-9204
Forum about error with DNA Master regarding FTP Connection (assuming Pitt server is not down at the time): https://seaphages.org/forums/topic/5155/?page=1#post-8693
Forum about errors primarily with DNA Master once downloaded: https://seaphages.org/forums/topic/5109/
How to resize Windows/fix seamless mode: Based off the SeaPhages forum, you need the “auto-resize guest display” to be checked from the view tab at the top of your Mac while in the Windows emulator. If it is checked, but it is greyed out (from the linuxquestions), click the command (host button) and “L” button combination a few times until the screen resizes

https://seaphages.org/forums/topic/5109/

Forum about using WINE instead of VirtualBox: https://seaphages.org/forums/topic/17/
Also, this forum has some general information: https://seaphages.org/forums/topic/4761/
Forum about M1 Macs “unsupported architecture” https://seaphages.org/forums/topic/5256/. Parallels costs money in a yearly subscription. Other workarounds still being investigated as of Jan. 2022 on this forum
Troubleshooting: if you start your DNA Master file and it says, “No permission”, you have the file saved in a spot that you do not have access to. A work-around is every time you start DNA Master, you will have to right-click on the start icon and click “run as administrator.” Also, can follow this forum to set the icon properly: https://seaphages.org/forums/topic/78/?page=1#post-506
Setting DNA Master Preferences: https://seaphagesbioinformatics.helpdocsonline.com/article-66

Thanks, Christina!
–Dan

Posted in: DNA Master → Common DNA Master on Mac Troubleshooting

Link to this post \| posted 27 Jan, 2022 18:33
DanRussell	Hi all, Christina Spencer from LeTourneau wanted to share a fix for the follow error she encountered helping students set up VirtualBox on Macs to install DNA Master. If you try to launch your new VM and get this error (kernel driver not installed): You can follow the steps at this website: https://appuals.com/kernal-driver-not-installed-rc-1908-error-mac/ The subsection “Allow Oracle Certificate through System Preferences” has step-by-step instructions that can be followed. This error is caused by your Mac not approving the program to run and you need to give the program permission. Follow your system prompts about giving permissions to the VirtualBox/Windows if prompted to proceed with next steps. Thanks, Christina! –Dan

Link to this post | posted 27 Jan, 2022 18:33

DanRussell

Hi all,

Christina Spencer from LeTourneau wanted to share a fix for the follow error she encountered helping students set up VirtualBox on Macs to install DNA Master. If you try to launch your new VM and get this error (kernel driver not installed):

You can follow the steps at this website: https://appuals.com/kernal-driver-not-installed-rc-1908-error-mac/

The subsection “Allow Oracle Certificate through System Preferences” has step-by-step instructions that can be followed. This error is caused by your Mac not approving the program to run and you need to give the program permission.

Follow your system prompts about giving permissions to the VirtualBox/Windows if prompted to proceed with next steps.

Thanks, Christina!
–Dan

Posted in: DNA Master → VirtualBox error: Kernel driver not installed

Link to this post \| posted 10 Jan, 2022 20:22
DanRussell	I would try increasing the base memory for the VM to like 4096 Mb or even higher. 2 Gb (your current setting) is quite low for Windows to be able to run, so increasing that should help. It won't necessarily use all the memory you allocate, but it's better to be higher. I also have 16 Gb on my Mac, and my Windows 10 VM has 6140 Mb of memory allocated. –Dan

Posted in: DNA Master → DNA Master and Windows 10

Link to this post \| posted 10 Jan, 2022 16:56
DanRussell	Hey Nancy, Not sure, but it sounds like your computer might be struggling to run the virtual Windows. What's the base RAM of your Mac computer? And how much memory and video memory did you allocate to the Windows VM? –Dan

Posted in: DNA Master → DNA Master and Windows 10

Link to this post \| posted 17 Dec, 2021 19:30
DanRussell	mary.preuss I haven’t even started a DNAM file. This happened immediately when I started the program for the first time on this new computer. For future folks: Mary and I did some troubleshooting offline, and it turned out it was the folder name she'd installed DNA Master into that was giving her issues. She installed DNA Master into a folder that had an apostrophe in its name, like "Mary's Stuff", and that apostrophe was confusing DNA Master's ability to correctly locate directories and save files. So don't use any unusual characters in the names of your filesystem, stick to letters and numbers only! I've updated the DNA Master installation instructions to reflect this. –Dan

Link to this post | posted 17 Dec, 2021 19:30

DanRussell

mary.preuss
I haven’t even started a DNAM file. This happened immediately when I started the program for the first time on this new computer.

For future folks: Mary and I did some troubleshooting offline, and it turned out it was the folder name she'd installed DNA Master into that was giving her issues. She installed DNA Master into a folder that had an apostrophe in its name, like "Mary's Stuff", and that apostrophe was confusing DNA Master's ability to correctly locate directories and save files.

So don't use any unusual characters in the names of your filesystem, stick to letters and numbers only!

I've updated the DNA Master installation instructions to reflect this.

–Dan

Posted in: DNA Master → TbQueries

Link to this post \| posted 17 Nov, 2021 16:24
DanRussell	Hi Kyle, Very interesting stuff! We have some Nanopore experience as well, but I'm pretty wary on its readiness to be a one-technology phage-sequencing option. In our most recent runs using a previously-sequenced (known) phage, single reads are around 89% accurate, but even high-coverage assemblies are still only around 98-99% accurate. Obviously, that means than 1 in every 50-100 bases would be wrong or gapped—even after lots of coverage—and that's not good enough to consider a phage "sequenced" or proceed with annotation. (Side note: many of the remaining errors were 1-2 base insertions/deletions, so they'd definitely throw a wrench in annotation.) That said, technologies improve over time, as does the software to make sense of their raw data. To really feel confident that an only-Nanopore-sequenced phage genome is reliable, we'd need to do several phages with known sequences and compare the Nano output to the reference. Chris actually did this with PacBio sequencing a bunch of years ago, and convinced me that when using the proper type of PacBio reads with enough coverage, you could trust a final sequence that came out of PacBio. You're right that, while Illumina-Nano hybrid assemblies have been great for bacterial sequencing, they're overkill for phages. Since almost all phages assemble fine with Illumina reads only, the Nanopore isn't necessary. But that doesn't mean it can't have a use in phage research or a SEA-PHAGES classroom. For example, it's probably economically feasible (and cool) for students to each get a little bit of Nanopore data for their phages, and then you could use that to decide which ones to send for Illumina sequencing, or add a Cluster to the phage's profile. We'll be talking about this stuff more at the next virtual faculty meeting! I think it's slated for Dec 17th, hopefully you'll be free. Quick question: which Nanopore library prep kit did you use for you phage sequencing? –Dan

Link to this post | posted 17 Nov, 2021 16:24

DanRussell

Hi Kyle,

Very interesting stuff! We have some Nanopore experience as well, but I'm pretty wary on its readiness to be a one-technology phage-sequencing option. In our most recent runs using a previously-sequenced (known) phage, single reads are around 89% accurate, but even high-coverage assemblies are still only around 98-99% accurate. Obviously, that means than 1 in every 50-100 bases would be wrong or gapped—even after lots of coverage—and that's not good enough to consider a phage "sequenced" or proceed with annotation.

(Side note: many of the remaining errors were 1-2 base insertions/deletions, so they'd definitely throw a wrench in annotation.)

That said, technologies improve over time, as does the software to make sense of their raw data. To really feel confident that an only-Nanopore-sequenced phage genome is reliable, we'd need to do several phages with known sequences and compare the Nano output to the reference. Chris actually did this with PacBio sequencing a bunch of years ago, and convinced me that when using the proper type of PacBio reads with enough coverage, you could trust a final sequence that came out of PacBio.

You're right that, while Illumina-Nano hybrid assemblies have been great for bacterial sequencing, they're overkill for phages. Since almost all phages assemble fine with Illumina reads only, the Nanopore isn't necessary. But that doesn't mean it can't have a use in phage research or a SEA-PHAGES classroom. For example, it's probably economically feasible (and cool) for students to each get a little bit of Nanopore data for their phages, and then you could use that to decide which ones to send for Illumina sequencing, or add a Cluster to the phage's profile.

We'll be talking about this stuff more at the next virtual faculty meeting! I think it's slated for Dec 17th, hopefully you'll be free.

Quick question: which Nanopore library prep kit did you use for you phage sequencing?

–Dan

Posted in: Sequencing, Assembling, and Finishing Genomes → Nanopore

Link to this post \| posted 21 Sep, 2021 20:04
DanRussell	Hi Steve, Jeffrey has made a couple of changes that should help: Moved the DNA Master install file to an HTTP address instead of an FTP address to make the initial download easier Changed the default FTP mode within DNA Master from Active to Passive The first will help with downloading the installer, while the second should make the first couple of updates work on most systems. That said, the usual caveats still apply: run as Admin, make sure preferences are correct, etc. Not sure why yours suddenly stopped working, but mine seems to be okay. –Dan Edited 21 Sep, 2021 20:05

Link to this post | posted 21 Sep, 2021 20:04

DanRussell

Hi Steve,

Jeffrey has made a couple of changes that should help:

Moved the DNA Master install file to an HTTP address instead of an FTP address to make the initial download easier
Changed the default FTP mode within DNA Master from Active to Passive

The first will help with downloading the installer, while the second should make the first couple of updates work on most systems. That said, the usual caveats still apply: run as Admin, make sure preferences are correct, etc. Not sure why yours suddenly stopped working, but mine seems to be okay.

–Dan

Edited 21 Sep, 2021 20:05

Posted in: DNA Master → DNA master server down?

Link to this post \| posted 12 Aug, 2021 19:19
DanRussell	byrumc@cofc.edu Quick question…During assembly of the genomes, is there a program used to trim the reads or is there no need to trim the 150-bp single end reads before looking at them in Newbler? Thanks! Christine Hi Christine, We usually don't bother trimming the reads when doing phage assembly since it is usually fairly straightforward. So raw reads are totally fine. We do use skewer to trim reads for our bacterial assemblies, partly because those are often 300-base reads and have lower quality more frequently towards their ends. https://github.com/relipmoc/skewer –Dan

Link to this post | posted 12 Aug, 2021 19:19

DanRussell

byrumc@cofc.edu
Quick question…During assembly of the genomes, is there a program used to trim the reads or is there no need to trim the 150-bp single end reads before looking at them in Newbler? Thanks!

Christine

Hi Christine,

We usually don't bother trimming the reads when doing phage assembly since it is usually fairly straightforward. So raw reads are totally fine.

We do use skewer to trim reads for our bacterial assemblies, partly because those are often 300-base reads and have lower quality more frequently towards their ends.

https://github.com/relipmoc/skewer

–Dan

Posted in: Newbler → Getting Started with Phage Assembly

Link to this post \| posted 15 Jul, 2021 14:25
DanRussell	Hey Evan, You make a good case as to why this one merits a check as a potential sequencing error, and it has some of those red flags (different from similar genomes, breaks a gene). But I just checked the sequencing data and see this: The base in question is a couple to the right of the green line, and the "A" called there is really strongly supported with no conflicting reads. So it's a real biological thing! –Dan

Link to this post | posted 15 Jul, 2021 14:25

DanRussell

Hey Evan,

You make a good case as to why this one merits a check as a potential sequencing error, and it has some of those red flags (different from similar genomes, breaks a gene). But I just checked the sequencing data and see this:

The base in question is a couple to the right of the green line, and the "A" called there is really strongly supported with no conflicting reads. So it's a real biological thing!

–Dan

Posted in: Annotation → Is this a sequencing error?

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