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Link to this post \| posted 01 Mar, 2022 17:34
DanRussell	uOttawaPHAGE 1. Do you use the NEB ligation kit? NEBNext companion module for Oxford nanowire technologies ligation sequencing https://international.neb.com/products/e7180-nebnext-companion-module-for-oxford-nanopore-technologies-ligation-sequencing#Product%20Information E7180S (24 rxns) Yes, we use that kit as called for in the protocol. uOttawaPHAGE 2. We were going to avoid the Q20 kits until they are more widely used. So I'm assuming we need this kit from oxford nanopore: https://store.nanoporetech.com/ligation-sequencing-kit.html 3. And these barcodes for up to 24 samples: https://store.nanoporetech.com/native-barcoding-expansion-1-12.html https://store.nanoporetech.com/native-barcoding-expansion-13-24.html Yep, those are the ones we use though we've never done more than 8 at a time. uOttawaPHAGE 4. Similar question as on my MiSeq post: has anyone tried using these kits at half volume? Haven't tried this. uOttawaPHAGE 5. Will they ship the first flow cell separately from the device? Dan mentioned flow cell aging is an issue, but we may have to spend the money we have before we have the libraries are ready…. I honestly don't remember re: the starter pack, but in general you can ask for delayed ship dates on the Nanopore website if you don't want them to ship immediately. uOttawaPHAGE 6. Someone mentioned to me the flow cells can be reused, which seemed confusing given what Dan had mentioned about old flow cells. If they are cleaned and reused in a short time, will that work? This would be a great feature for using it in our course. Yes, flow cells sort of have a "useful lifetime" in hours of sequencing. They usually say the flowcells can sequence for 72 hours. So you can stop after 4 hours, wash, and then do a different run later. But there's a substantial decline over time (both active sequencing time and sedentary fridge time)in the number of available pores, so you might have to sequence the second run for 12 hours to get the same amount of sequence as the first run got in 4 hours. A third run might need 48 hours. And so on. So you can't really look at that "72 hours" as like 6 equal 12-hour runs. There's also some carryover in barcodes from one run to the next. Though it's not a ton, it can be confounding if you don't have a ton of reads. Good luck! –Dan

Link to this post | posted 01 Mar, 2022 17:34

uOttawaPHAGE
1. Do you use the NEB ligation kit? NEBNext companion module for Oxford nanowire technologies ligation sequencing https://international.neb.com/products/e7180-nebnext-companion-module-for-oxford-nanopore-technologies-ligation-sequencing#Product%20Information
E7180S (24 rxns)

Yes, we use that kit as called for in the protocol.

uOttawaPHAGE
2. We were going to avoid the Q20 kits until they are more widely used. So I'm assuming we need this kit from oxford nanopore:

https://store.nanoporetech.com/ligation-sequencing-kit.html

3. And these barcodes for up to 24 samples:

https://store.nanoporetech.com/native-barcoding-expansion-1-12.html

https://store.nanoporetech.com/native-barcoding-expansion-13-24.html

Yep, those are the ones we use though we've never done more than 8 at a time.

uOttawaPHAGE
4. Similar question as on my MiSeq post: has anyone tried using these kits at half volume?

Haven't tried this.

uOttawaPHAGE
5. Will they ship the first flow cell separately from the device? Dan mentioned flow cell aging is an issue, but we may have to spend the money we have before we have the libraries are ready….

I honestly don't remember re: the starter pack, but in general you can ask for delayed ship dates on the Nanopore website if you don't want them to ship immediately.

uOttawaPHAGE
6. Someone mentioned to me the flow cells can be reused, which seemed confusing given what Dan had mentioned about old flow cells. If they are cleaned and reused in a short time, will that work? This would be a great feature for using it in our course.

Yes, flow cells sort of have a "useful lifetime" in hours of sequencing. They usually say the flowcells can sequence for 72 hours. So you can stop after 4 hours, wash, and then do a different run later. But there's a substantial decline over time (both active sequencing time and sedentary fridge time)in the number of available pores, so you might have to sequence the second run for 12 hours to get the same amount of sequence as the first run got in 4 hours. A third run might need 48 hours. And so on. So you can't really look at that "72 hours" as like 6 equal 12-hour runs.

There's also some carryover in barcodes from one run to the next. Though it's not a ton, it can be confounding if you don't have a ton of reads.

Good luck!
–Dan

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

Link to this post \| posted 22 Feb, 2022 20:52
DanRussell	Hi SEA-PHAGES community, Phage-obsessed students who are about to graduate or who have recently graduated might be interested in this opportunity! It's a phage therapy research position at the FDA. https://www.zintellect.com/Opportunity/Details/FDA-CBER-2022-18 Good luck, –Dan

Posted in: General Message Board → FDA Phage Therapy Position for Recent Grad

Link to this post \| posted 08 Feb, 2022 15:53
DanRussell	amaya debbie Chris, I am not sure what you are answering but do your DNA master preference panes look like this? Debbie, None of my students DNAMaster Preferences page looked like that -like the one on the manual and the one you have posted here. They were missing the bottom part (with the box checked) and the rest of the menu. They've installed DNAMaster from the link, and have updated and restarted, so I am not sure what else to do to get them to match the file from the guide. They also didn't have a Secure Connections tab (under Internet). One of them got the tRNAs autoannotated, but glimmer failure otherwise, and I think that that is related to these pages not being the same. I've sent them my auntoannotation for now and we are meeting again tomorrow so hopefully the y can do other functions of DNAMaster, but so far no autoannotations from them. Hi Amaya, Are you sure they've updated DNA Master? What build number are they running? Because if they've successfully updated, they should have the Secure Connections tab. If they haven't, then we need to figure out what's preventing them from udpating. –Dan

Link to this post | posted 08 Feb, 2022 15:53

DanRussell

amaya
debbie
Chris,
I am not sure what you are answering but do your DNA master preference panes look like this?

Debbie,
None of my students DNAMaster Preferences page looked like that -like the one on the manual and the one you have posted here. They were missing the bottom part (with the box checked) and the rest of the menu.

They've installed DNAMaster from the link, and have updated and restarted, so I am not sure what else to do to get them to match the file from the guide. They also didn't have a Secure Connections tab (under Internet). One of them got the tRNAs autoannotated, but glimmer failure otherwise, and I think that that is related to these pages not being the same. I've sent them my auntoannotation for now and we are meeting again tomorrow so hopefully the y can do other functions of DNAMaster, but so far no autoannotations from them.

Hi Amaya,

Are you sure they've updated DNA Master? What build number are they running? Because if they've successfully updated, they should have the Secure Connections tab. If they haven't, then we need to figure out what's preventing them from udpating.

–Dan

Posted in: DNA Master → Auto-annotation fix for fall 2017 and later

Link to this post \| posted 07 Feb, 2022 15:26
DanRussell	Hi all, Please see the message below from Eric Miller about a summer research opportunity for undergrads this year. –Dan Hello all, I hope you are doing well. We are recruiting undergraduates for the forthcoming summer 2022 REEU BeeMORE (Bees and Microbes) program at NC State University. Our goal is to recruit as many students underrepresented in agriculture and science as possible; our participants are selected based on their motivation to learn and to make the most of our 9-week program. Undergraduates in their second or third year are preferred. Here is the link to our home page: https://harvest.cals.ncsu.edu/beemore/ On the web page there is a recent video describing the program, a flyer suitable for printing or sharing electronically, and a link to the brief application procedure. https://youtu.be/hKR5G7YoASg Please share broadly! Thank you for helping get the word out, to your students and to your colleagues. Post it on one of your web pages citing summer opportunities! Thank you and all the best, Eric Eric S. Miller, Ph.D. Professor of Microbiology Department of Plant & Microbial Biology North Carolina State University Raleigh, NC 27695-7615

Link to this post | posted 07 Feb, 2022 15:26

DanRussell

Hi all,

Please see the message below from Eric Miller about a summer research opportunity for undergrads this year.

–Dan
Hello all,

I hope you are doing well.

We are recruiting undergraduates for the forthcoming summer 2022 REEU BeeMORE (Bees and Microbes) program at NC State University. Our goal is to recruit as many students underrepresented in agriculture and science as possible; our participants are selected based on their motivation to learn and to make the most of our 9-week program. Undergraduates in their second or third year are preferred.

Here is the link to our home page: https://harvest.cals.ncsu.edu/beemore/

On the web page there is a recent video describing the program, a flyer suitable for printing or sharing electronically, and a link to the brief application procedure.
https://youtu.be/hKR5G7YoASg

Please share broadly! Thank you for helping get the word out, to your students and to your colleagues. Post it on one of your web pages citing summer opportunities!

Thank you and all the best,
Eric

Eric S. Miller, Ph.D.
Professor of Microbiology
Department of Plant & Microbial Biology
North Carolina State University
Raleigh, NC 27695-7615

Posted in: General Message Board → NC State Summer 2022 BeeMORE Program for Undergrads

Link to this post \| posted 01 Feb, 2022 21:57
DanRussell	Hi Steve, There's a forum thread that talks about this error, but apparently the link to the fix isn't working anymore. https://seaphages.org/forums/topic/4436/ But this should be basically the same thing. Maybe try installing one of the Visual C++ packages here and giving it a shot? https://docs.microsoft.com/en-US/cpp/windows/latest-supported-vc-redist?view=msvc-170 –Dan

Posted in: DNA Master → Could not load SSL library

Link to this post \| posted 31 Jan, 2022 20:33
DanRussell	Hi Adam, Some answers below. 1. Is this the library kit? NEB #E7805S Any opinions about using NEB SPRIselect vs. AMPure XP beads? Yes, that's one. Any of the NEBNext Ultra II FS kits will work. The "FS" part is for shearing your DNA. 2. Do you use dual or single indexed adaptors/barcodes? If single indexed, do you purchase a 96 barcode kit (NEB E7335L)? Or are there 48 barcode kits? Or can this be DIY? When we do our typical runs of 48 phages, we use dual indexing with this barcode kit (#E7600S): https://www.neb.com/products/e7600-nebnext-multiplex-oligos-for-illumina-dual-index-primers-set-1#Product%20Information That one gives 96 possible dual-indexed combos. When we do smaller runs, we sometimes use single-indexing with a kit like this (#E7335S): https://www.neb.com/products/e7335-nebnext-multiplex-oligos-for-illumina-index-primers-set-1 You can do it DIY, some people do, but not us! 3. For library construction, can reactions be done in half volume? We tried this for a IonTorrent library and it worked fine. Obviously not the manufacturer's instructions, but it would save us quite a bit of money. We've never tried, but FYI our output library concentration from the current workflow is often not too far above the "minimum" 4nM required for proper MiSeq loading, so it's possible you'd be too low. Also, some of the volumes would be quite low, like resuspending beads in 8.5 µl of buffer, which could be a bit tricky. 4. Do you use the Illumina v2 reagent kit? We use 150-cycle v3 Illumina kits. You can instruct the MiSeq to do 1x150bp reads. (Rather than paired end reads.) 5. Any other consumables needed? Is there a distinct wash kit or does it come with the reagent kit? Check the NEB and Illumina protocols for specifics about what's needed, but there's no too much that's not standard lab fare. Washing the MiSeq is just done with Tween diluted in water, so you'll need the right Tween, and water, but no kit necessary. 6. I believe Pitt uses a Qubit to assess the library. We have access to one, but I've not used it, Are there special reagents needed? Or specific settings? Yes, you'll need a Qubit Broad Range DNA Kit to quantify DNA using a Qubit. We use 1 µl of sample and 199 µl of QWS. You then just have to tell the Qubit itself what you used, and it'll tell you your stock concentration. 7. Any details on the MiSeq settings? Are you running 2X150bp reads? I will have help from someone who has sequenced yeast genomes, so his settings may be different (if there are other settings….). As mentioned above, we use 150-cycle v3 kits, set to 1x150bp reads. When making a sample sheet, we select "FASTQ only". 8. Someone sent me a DIY protocol for the AMPure beads. Any experience making them? We'll purchase some for this project, but long-term we would consider this. See attached. Definitely haven't done that. You can get the above-referenced library prep kits with beads included, but I think basically any of the purification beads are okay as long as they work. In general, it sounds like you're trying to keep library prep costs low which is understandable. Perhaps even better than half-volume reactions would be to combine two phages you know—for example, by restriction digest—belong to different clusters. Then you'd get two complete phage sequences out of each library prep. There's no doubt that people use a variety of DIY and tweaks, but we've found that the cost per library from NEB is < $40 and we're more concerned with throughput and success %, so we haven't experimented. Good luck! –Dan Edited 31 Jan, 2022 20:35

Link to this post | posted 31 Jan, 2022 20:33

DanRussell

Hi Adam,

Some answers below.

1. Is this the library kit? NEB #E7805S Any opinions about using NEB SPRIselect vs. AMPure XP beads?

Yes, that's one. Any of the NEBNext Ultra II FS kits will work. The "FS" part is for shearing your DNA.

2. Do you use dual or single indexed adaptors/barcodes? If single indexed, do you purchase a 96 barcode kit (NEB E7335L)? Or are there 48 barcode kits? Or can this be DIY?

When we do our typical runs of 48 phages, we use dual indexing with this barcode kit (#E7600S): https://www.neb.com/products/e7600-nebnext-multiplex-oligos-for-illumina-dual-index-primers-set-1#Product%20Information

That one gives 96 possible dual-indexed combos. When we do smaller runs, we sometimes use single-indexing with a kit like this (#E7335S): https://www.neb.com/products/e7335-nebnext-multiplex-oligos-for-illumina-index-primers-set-1

You can do it DIY, some people do, but not us!

3. For library construction, can reactions be done in half volume? We tried this for a IonTorrent library and it worked fine. Obviously not the manufacturer's instructions, but it would save us quite a bit of money.

We've never tried, but FYI our output library concentration from the current workflow is often not too far above the "minimum" 4nM required for proper MiSeq loading, so it's possible you'd be too low. Also, some of the volumes would be quite low, like resuspending beads in 8.5 µl of buffer, which could be a bit tricky.

4. Do you use the Illumina v2 reagent kit?

We use 150-cycle v3 Illumina kits. You can instruct the MiSeq to do 1x150bp reads. (Rather than paired end reads.)

5. Any other consumables needed? Is there a distinct wash kit or does it come with the reagent kit?

Check the NEB and Illumina protocols for specifics about what's needed, but there's no too much that's not standard lab fare. Washing the MiSeq is just done with Tween diluted in water, so you'll need the right Tween, and water, but no kit necessary.

6. I believe Pitt uses a Qubit to assess the library. We have access to one, but I've not used it, Are there special reagents needed? Or specific settings?

Yes, you'll need a Qubit Broad Range DNA Kit to quantify DNA using a Qubit. We use 1 µl of sample and 199 µl of QWS. You then just have to tell the Qubit itself what you used, and it'll tell you your stock concentration.

7. Any details on the MiSeq settings? Are you running 2X150bp reads? I will have help from someone who has sequenced yeast genomes, so his settings may be different (if there are other settings….).

As mentioned above, we use 150-cycle v3 kits, set to 1x150bp reads. When making a sample sheet, we select "FASTQ only".

8. Someone sent me a DIY protocol for the AMPure beads. Any experience making them? We'll purchase some for this project, but long-term we would consider this. See attached.

Definitely haven't done that. You can get the above-referenced library prep kits with beads included, but I think basically any of the purification beads are okay as long as they work.

In general, it sounds like you're trying to keep library prep costs low which is understandable. Perhaps even better than half-volume reactions would be to combine two phages you know—for example, by restriction digest—belong to different clusters. Then you'd get two complete phage sequences out of each library prep.

There's no doubt that people use a variety of DIY and tweaks, but we've found that the cost per library from NEB is < $40 and we're more concerned with throughput and success %, so we haven't experimented.

Good luck!
–Dan

Edited 31 Jan, 2022 20:35

Posted in: Sequencing, Assembling, and Finishing Genomes → MiSeq protocol and info

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

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All posts created by DanRussell