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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27022057 - 11/04/20 08:14 PM (5 months, 27 days ago)

Here's the sequence analysis video I made the other day.



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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27023013 - 11/05/20 12:32 PM (5 months, 26 days ago)

Wow, very cool! So thankful that you took the time to create this video and continue to educate us. 🙏 Seeing an example that is so close to home really helps the knowledge to connect.

Do you mind if I share the video on my socials?

I'm working on a GMO yeast to produce baeocystin and psilocybin. I set a goal to have a prototype in the next 90 days. Any interest in testing it or providing feedback? You've already done so much so I won't be offended if you stay on the sidelines!

Thanks again man.


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@EverymanBio | EverymanBio Podcast | YouTube


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27023482 - 11/05/20 05:33 PM (5 months, 26 days ago)

Go ahead and share the video.

It would be fun to test your yeast.  Are those separate strains to produce baeocystin and psilocybin, or one strain does both?    Would be really neat if they were separate.


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27023565 - 11/05/20 06:21 PM (5 months, 26 days ago)

There are five genes that define the metabolic process of converting glucose to psilocybin. Baeocystin is produced as the final precursor the last gene uses to produce psilocybin. The plan is to omit the final gene from the plasmid, mostly for legal reasons, but also because it should mean I generate an abundance of baeocystin and norbaocystin since there will be nothing to metabolize it. Adding the final gene to produce psilocybin will be trivial at that point.

Lot's of work to get there but it's currently 100% of where I'm spending my time. I'm pretty excited to see if I can get a pure sample of baocystin, try it and give it out for research.


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27031512 - 11/10/20 01:58 PM (5 months, 21 days ago)

How long does it usually take for a new Genbank sequence to process and be approved? I published a sequence to GenBank yesterday and it's still processing.

I'm wondering if it's delayed because I put Penicillium labradorum as the locus and source origin but it isn't in the NCBI taxonomy database yet.


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27032283 - 11/10/20 10:32 PM (5 months, 21 days ago)

Quote:

PTreeDish said:
How long does it usually take for a new Genbank sequence to process and be approved? I published a sequence to GenBank yesterday and it's still processing.

I'm wondering if it's delayed because I put Penicillium labradorum as the locus and source origin but it isn't in the NCBI taxonomy database yet.




It takes one week.

They'll add it to the database.


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27032410 - 11/11/20 12:22 AM (5 months, 21 days ago)

Cool. I was just notified it finished processing. Says it'll be live on 11/15. When it's live, would you mind checking the record to ensure I added the appropriate metadata to be useful? Accession AMW241166


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27043996 - 11/17/20 04:41 PM (5 months, 14 days ago)

My GenBank submission is live.

Sadly, the baeocystin yeast project isn't feasible for a one-man team. It would cost over $10K just in gene synthesis and plasmid construction. :/ I'll need to find a simpler project to learn genetic engineering.


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OfflineBlueMushies
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27061774 - 11/28/20 05:06 PM (5 months, 3 days ago)

I’m really interested in this and was wondering if you guys and point me in the direction on where to start and any equipment I can get my hands on to start learning will be great. Keep up the awesome work you guys truly inspiring.


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: BlueMushies]
    #27074401 - 12/06/20 01:45 AM (4 months, 27 days ago)

Quote:

BlueMushies said:
I’m really interested in this and was wondering if you guys and point me in the direction on where to start and any equipment I can get my hands on to start learning will be great. Keep up the awesome work you guys truly inspiring.





https://wiki.counterculturelabs.org/wiki/DNA_sequencing


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OfflineBlueMushies
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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27075224 - 12/06/20 02:37 PM (4 months, 26 days ago)

Thank you Alan


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Re: DNA barcoding class / interview with Adam Haritan [Re: BlueMushies]
    #27095667 - 12/18/20 01:07 PM (4 months, 14 days ago)

Hey y'all, Just got some results back from Genewiz and half of my samples came back with the issue "no priming", while the rest look great. Just wondering where to go from here for troubleshooting? Most of the genewiz troubleshooting suggestions seem to be based around issues with the primers used. I used the ITS1F and ITS4R primers from the Odin, which worked great for half my samples and seem to work for everyone else. My gel electrophoresis results seem fine. Should I mess with annealing temperature/time? Or is it more likely issues with my extraction process? I used the protocol by Sigrid Jakob (https://docs.google.com/document/d/13B9OSE_ar_vWWZnHZegr2FROnMak78qHZxEZXc1E9jk/edit) Any suggestions/feedback are appreciated. Thanks and happy holidays


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: Jawn876]
    #27096707 - 12/18/20 11:20 PM (4 months, 14 days ago)

I've done ~25+ sequences now and I have about a 50% success rate. I do wonder how typical this is. I've not talked to a single person than claims a near perfect success rate.

You are welcome to see what all I've tried by looking at my sequencing data tracker:
https://docs.google.com/spreadsheets/d/1cVpw745ZRqTKRAINBVLIgbKZ4y4a-mXCbfuWa0cO9jw/edit#gid=0

It doesn't include my first 8 samples with MCLab which all failed. :smile:

Genewiz has a troubleshooting guide that covers the myriad of potential reasons for the "no priming" error here:
https://web.genewiz.com/hubfs/GA_Diagnosis_Sanger-sept19.pdf

I've found some of them difficult to test for, like DNA concentration, without additional equipment - most of which is out of my reach for a diyer working at-home. Like yourself, I've had good gels for every sample.

My understanding could be completely wrong here, but I think in the case of contaminations, where maybe you PCR'd more than one specie's ITS gene unintentionally, there appears to be only one band since bands from both organisms will be pretty close to the same size. I don't know of anyway to test for this ahead of time.

Do you know how to read the chromatographs? At some least some of my errors have been caused by contamination as evident by many overlapping base peaks.

Brittany at Genewiz has been incredible with helping me. She reruns a lot of my stuff multiple times and gives me discounts on retries. Unfortunately, I've not had a single retry ever work.

Happy Holidays!


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InvisibleJawn876
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27098369 - 12/19/20 09:50 PM (4 months, 13 days ago)

Quote:

I do wonder how typical this is. I've not talked to a single person than claims a near perfect success rate.




Me too, seems we have about the same success rate. I wonder what the success rate is in professional laboratories.
Thanks for sharing your tracker, cool to see. And thanks for that genewiz link, it has different troubleshooting suggestions than the one I get linked to from them ( https://clims4.genewiz.com/Common/TroubleShooting/en-US/No%20Priming.pdf )

Quote:

I think in the case of contaminations, where maybe you PCR'd more than one specie's ITS gene unintentionally, there appears to be only one band since bands from both organisms will be pretty close to the same size.




Solid theory. I have been doing the process in front of my flowhood but contamination is most likely present. Im pretty certain that if I put the same sample tissue on an agar dish it would not be perfectly clean mycelium. Maybe I'll do a run of all mycelium cultures from clean dishes and see if the results are better. I've been using tissue samples from wild specimens.

I think I understand how to clean up a good chromatogram but I don't really know much about analyzing the bunk ones. I'll do some reading, any suggested resources are appreciated.

I'd really like to get a better success rate with this. Maybe this is a shitty perspective but it feels like when factoring in a 50% success rate, it basically doubles the price of success so its almost $20 per result. I'm really interested in this but I'm not sure how sustainable this is for my budget. However, I am confident a better success rate is possible. Still not really sure what to do differently but I'll keep trying and learning.

Thanks for your thoughts!


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: Jawn876]
    #27101229 - 12/21/20 06:40 PM (4 months, 11 days ago)

No priming is a common problem, and I don't know what causes it.  Genewiz offers free repeats, and sometimes that helps but not usually.  Also try sequencing it in the reverse direction with the ITS4 primer.

Sometimes it's a bad DNA purification at Genewiz, which they can redo, but when you order a repeat or additional sequencing, they use the same DNA template and don't usually redo the purification step, though they can if they think it might be an issue.

It's a good idea to email them and send them a photo of the gel and ask what went wrong.  They might be able to help, and at the very least they'll give you some free sequences on your next order.

Re-running PCR is a good idea, with the same or different primers.  You could switch out ITS1F for ITS1 or ITS3, and switch out ITS4 for ITS2 or ITS5 or ITS4B or TW13.

You could also try a different gene like LSU or TEF1 or beta-tubulin, though if there aren't many sequences in Genbank for your genus then you might not have data to compare against.  It's always a good idea to check what's in Genbank before deciding what gene you want - for basidiomycetes, ITS is the right answer 90% of the time.


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OfflineeLeSDenesS
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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27101266 - 12/21/20 07:01 PM (4 months, 11 days ago)

Hey Alan,

You mentioned in your tutorial that genewiz offers sequencing after PCR. Does minION sequencer does the same thing?


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: eLeSDenes]
    #27101938 - 12/22/20 04:43 AM (4 months, 11 days ago)

Quote:

eLeSDenes said:
You mentioned in your tutorial that genewiz offers sequencing after PCR. Does minION sequencer does the same thing?




Sort of - A Minion run is expensive, $1000 if you use the whole flow cell.  Genewiz charges $8 to purify and sequence a PCR product.

The sequencing methodology is also different - the Sanger sequencing that Genewiz offers gives you an average of all of the copies of the DNA in the sample.  Minion reads DNA one strand at a time, so you get the individual data instead of the average of all data.  The data is usually similar, perhaps the same, it will be interesting to see how different it is. 

It is possible to run PCR with tags the attach to each strand of DNA, then pool a lot of PCR runs and sequence them all at once with the Minion, then use the tags to separate the data into each individual sample.  I am not sure how the price of doing that would compare to Sanger runs, it depends on how long you run it (how much of the flow cell you use up).


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27103089 - 12/22/20 10:31 PM (4 months, 10 days ago)

Thanks for taking the time to answer. I though that the consumables for the minion are much cheaper - a single flow sell is $1000 as you said. I guess I just have to stick with sending the samples in for sequencing.


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27181373 - 02/01/21 06:48 PM (3 months, 13 hours ago)

Hey Alan, I'm just finishing up another batch of 20 sequences and I've come across a few use-cases with using BLAST that have me stumped. I was hoping you might lend a hand.

The first one involves a mold I captured on an unidentified polypore. Here is the trace file:
https://github.com/EverymanBio/chromatograms/blob/main/trace_files/batch_3/16_dark.spider.web.mold-ITS1-F.ab1

The sequenced I BLASTED (lowercase are my edits):

>16_dark.spider.web.mold-ITS1-F
gtTTNCGTaGTGACCTGCGGAAGGAtCATTACCGAGTGAGGACCCTCTGGGTCCAACCTCCCACCCGTGTTTATCGTACCTTGTTGCTTCGGCGGGCCCGCCGAAGACACCTGTGAACGCTGTATGAAGATTGCAGTCTGAGCGACAAGCTAAATTTGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCACGGCTTGTGTGTTGGGCCCTCGTCCCCCGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTCGTCACCCGCTCTGCAGGCCCGGCCGGCGCCTGCCGACACCATCAATCTTTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATAtca

The closest match is 99.07% to Penicillium rubidurum with what look like very clear differences between the top matches. What should I do in this case?

I have another sample just like this, matches 99% to Sistotrema sernanderi but with very clear single nucleotide differences.

Also, what do you when you have multiple 100% matches with different stated species in the results?

Finally, I have another one where it's not super clear in the trace file if I should have a single A or double AA in one place and having the AA would greatly improve the number of identical matches, even though there are also 100% matches with single A. What do you do here?

Thanks!


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Edited by PTreeDish (02/01/21 09:56 PM)


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27181887 - 02/02/21 12:09 AM (3 months, 8 hours ago)

Here's the type collection sequence of Penicillium parvum:  https://www.ncbi.nlm.nih.gov/nuccore/NR_121241.1

Looks like your sequence is 4 bases away from that, ignore the mismatch where it's just a different number of repeating T's, as Sanger often has issues sequencing repeated bases.

It looks like some of the Penicillium rubidurum and P. parvum sequences in Genbank aren't accurately named, look for the sequences that say "from TYPE material". 

You also may want to check additional gene regions like LSU and beta-tubulin, you can check to see which genes of the type collection were sequenced.  If you really care that much.  If it's not critical you could just call it Penicillium parvum group or something like that.

Sistotrema is in the Hydnaceae so the Sistotrema sequence that you are matching is misnamed, probably a Sistotrema that had Penicillium growing on it.  Sometimes when I see these I email the sequence authors and let them know that they could update their record.  Often they do bulk uploads and contaminates like this slip through the cracks.

When you have multiple 100% matches to different species, you could look into the metadata of the sequences and see which ones you trust most.  Some might be from researchers actually studying that group and others from hard partying grad students who aren't paying a lot of attention to what they are uploading.  Getting sequences from reliable taxonomic papers is a good idea.  If you are interested in a certain genus you could build a FASTA file with sequences you have vetted so you don't have to sift through all of the misnamed stuff.   

Don't put too much stock in those names, they are helpful pointers but they are just suggestions for the most part.  Even sequences uploaded with the wrong name are useful as you can see where else in the world your organism occurs and get an idea of how much the sequence of a certain species can vary.

Sometimes the gene you are using isn't enough to ID to species - like with Fusarium ITS will get you just to group and you need both ITS and TEF1 to get to species.


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