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Recent Activity
All posts created by DanRussell
| Link to this post | posted 18 Jan, 2023 16:29 | |
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Hi Alison, What kinds of computers do your students who are getting the error have? –Dan |
| Link to this post | posted 09 Nov, 2022 15:20 | |
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Hi Adam, 1. Yes, we use the standard Illumina protocol for denaturing/loading. The BioAnalyzer gives us numbers to adjust our starting pool to 4 nM. 2. Yes, we denature 5 µl of the 4nM pool. 3. We pool before we quantify, so we just take 5 µl of the pool. In your case, you'll need to pool libraries if you haven't done so. I'd recommend doing it in larger volumes than 0.25 µl though to avoid pipetting inaccuracy. If you've already normalized all the libraries @ 4 nM, then just take a microliter of each to make a 4nM pool, then take 5 µl out from that pool. (You should have plenty of extra library.) 4. We use the NEB Ultra II FS kit starting with 400 ng of input DNA. But other factors can determine final yield as well. For example, I think we only do 3 PCR cycles during the PCR step, but more cycles will get you more library in the end. Some libraries we've prepped are > 200 nM, others are only 5-10 nM. As long as you're above 4 nM, you should be fine. 5. Yes, we add between 3 and 6 µls of the phiX control before loading the sample. 6. Don't think so! Good luck, –Dan |
| Link to this post | posted 27 May, 2022 17:22 | |
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Hi Fernando, I'd recommend shooting for 100X coverage on your phage genomes. It might be a little higher or lower, but that's generally a decent target that's also not going to cost too much per phage. If you take an "average" phage genome of 70kb in length, that means you need ~7,000,000 bp of sequence to get to that coverage level. If the reads you're doing are 150 bases long, then: 7,000,000/150 = ~47,000 reads per genome Obviously that'll change depending on the size of the genome (often unknown!) and the length of the reads, so we usually shoot for ~100,000 reads per phage genome, though 50,000 will often be enough. As for protocols, we just use the standard protocol that comes with our library prep kit: https://www.neb.com/nebnext-ultra-ii-fns-dna/nebnext-ultra-ii-for-dna-library-prep After library prep, we use BioAnalyzer HS chips to check quantity/quality. Then the standard Illumina protocol to load the sequencer. We have a MiSeq, not a MiniSeq, so I don't have any specifics for the MiniSeq, but most of Illumina's sequencers these days are pretty easy to use. Take care and good luck! –Dan |
| Link to this post | posted 06 Apr, 2022 16:50 | |
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Hi Adam, Actually, they're not nearly nearly identical; there's only 32% amino acid identity between Arzan 58 and the next best match in the DB! (Which is Heisenberger 58 as of me writing this.) That's quite a lot of differences, and while it's probably reasonable to consider these homologs, they're still different enough that: 1. Phamerator's algorithm put them in different phams and 2. trying to make start calls on Arzan 58 based on Heisenberger 58 is not advisable. The AA sequences are 32% identical, but the nucleotide sequences show almost no similarity at all, so using a starterator report isn't a good idea in this situation, since SD sites and potential start codons have had lots of evolutionary time to change. This one you'll have to call on its own! –Dan |
Posted in: Annotation → Called an orpham but isn't?
| Link to this post | posted 01 Mar, 2022 17:34 | |
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uOttawaPHAGE Yes, we use that kit as called for in the protocol. uOttawaPHAGE Yep, those are the ones we use though we've never done more than 8 at a time. uOttawaPHAGE Haven't tried this. uOttawaPHAGE 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 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 22 Feb, 2022 20:52 | |
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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 |
| Link to this post | posted 08 Feb, 2022 15:53 | |
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amayadebbie 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 | |
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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 01 Feb, 2022 21:57 | |
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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 | |
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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 |