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OfflineAlan RockefellerM
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Re: Panaeolus phylogenetic tree [Re: wintersbefore]
    #17809974 - 02/15/13 08:44 PM (10 years, 11 months ago)

I think that the problem with your tree is that you chose everything that Genbank thought was similar, without vetting the sequences it gave you.  You got all of Psilocybe, and all sequences that are from Psilocybe but are misidentified as something else.

Try choosing a reasonable outgroup and retrieve the sequence for the outgroup by searching by name and choosing the most believable sequence available.

The problematic sequences are the ones that don't make any sense that are clustered with the ones that do make sense.


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Invisiblebloodworm
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Re: Panaeolus phylogenetic tree [Re: Alan Rockefeller]
    #17810335 - 02/15/13 09:46 PM (10 years, 11 months ago)

nice work Alan, very interesting...
however, the process seems seriously flawed (not on your end), imo.

:aliendance:
peace and love
bloodworm


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OfflineAlan RockefellerM
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Re: Panaeolus phylogenetic tree [Re: bloodworm]
    #17811017 - 02/15/13 11:56 PM (10 years, 11 months ago)

How is it flawed?


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Offlinewintersbefore
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Re: Panaeolus phylogenetic tree [Re: Alan Rockefeller]
    #17811703 - 02/16/13 04:35 AM (10 years, 11 months ago)

Quote:

Alan Rockefeller said:
How is it flawed?




Because my goal here was to generate a tree at finer scales than a published tree that I am using for reference in order to find P. cubensis' most closely related inactive relative.... and I was not able to do that because I would have already had to know that information in order to have a tree that makes sense.  For example, I would have to select sequences that I trust have not been misidentified to choose an "outgroup" (so I have to select something from another genus over, but how do I know what that is?!?) Is it Agrocybe praecox? If so, I've answered my question and I don't need the tree.


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OfflineAlan RockefellerM
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Re: Panaeolus phylogenetic tree [Re: wintersbefore]
    #17811712 - 02/16/13 04:40 AM (10 years, 11 months ago)

According to BLAST, Naucoria bohemica is the closest non-psychoactive mushroom to Psilocybe cubensis.

To see these results, go to this link, scroll down to the bottom of the page and click BLAST.

To get the best possible results I used a long cubensis sequence, 1997 base pairs.  But it appears that they tacked together its1, its2 and 28s.  Whether this actually gives the best results, I don't know.



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Offlinewintersbefore
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Re: Panaeolus phylogenetic tree [Re: Alan Rockefeller]
    #17811738 - 02/16/13 04:54 AM (10 years, 11 months ago)

Quote:

Alan Rockefeller said:
According to BLAST, Naucoria bohemica is the closest non-psychoactive mushroom to Psilocybe cubensis.



Would you say that Agrocybe praecox is the most closely related "common" mushroom? If not, what is (I have only seen a few pictures of Naucoria) so I guess I'd like to see, in a tree, how far away, at these finer scales, is A. praecox?

Quote:


To see these results, go to this link, scroll down to the bottom of the page and click BLAST.

To get the best possible results I used a long cubensis sequence, 1997 base pairs.  But it appears that they tacked together its1, its2 and 28s.  Whether this actually gives the best results, I don't know.







So are all those Lepistas, Tricholomas, and Hygrocybes misidentified?!? How does that even happen?? Those mushrooms don't look anything like a Psilocybe, and they are light spored!?!


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OfflineAlan RockefellerM
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Re: Panaeolus phylogenetic tree [Re: wintersbefore]
    #17811760 - 02/16/13 05:05 AM (10 years, 11 months ago)

Quote:

wintersbefore said:
Would you say that Agrocybe praecox is the most closely related "common" mushroom? If not, what is (I have only seen a few pictures of Naucoria) so I guess I'd like to see, in a tree, how far away, at these finer scales, is A. praecox?




I think that Agrocybe pediades is closer and also common, and that Panaeolus papilionaceus is closer still, and still common.


Quote:

So are all those Lepistas, Tricholomas, and Hygrocybes misidentified?!? How does that even happen?? Those mushrooms don't look anything like a Psilocybe, and they are light spored!?!




They tried to sequence a Psilocybe accidentally put the wrong dna in the well, an error caused by being distracted by people talking to them in the lab or forgetting which well is which due to smoking marijuana, or they didn't use a new pipette tip when they should have. 


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Offlinewintersbefore
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Re: Panaeolus phylogenetic tree [Re: Alan Rockefeller]
    #17811839 - 02/16/13 06:06 AM (10 years, 11 months ago)

Quote:

Alan Rockefeller said:

They tried to sequence a Psilocybe accidentally put the wrong dna in the well, an error caused by being distracted by people talking to them in the lab or forgetting which well is which due to smoking marijuana, or they didn't use a new pipette tip when they should have. 





While that does happen, it does not exactly explain what happened here.  It is possible that someone "was trying to sequence a Tricholoma" but accidentally sequenced a Psilocybe and when they got sequences back and the closest match was for Psilocybe, it should have been renamed before being submitted. Anyhow, all of those are errors in GenBank and there are far too many of them, much worse then wikipedia, because with wikipedia anyone can go in and fix the errors, but here, only the author's can right? Anyhow... unacceptable number of errors for this particular blast querry, I've not had as messy results with other things that I've querried...

Quote:

I think that Agrocybe pediades is closer and also common



Perfect! Thank you very much!


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InvisibleThe Lightning
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Re: Panaeolus phylogenetic tree [Re: wintersbefore]
    #18165417 - 04/25/13 09:11 AM (10 years, 9 months ago)

Alright. Alright. I have to stir up the Hornet's Nest for a second here.

I'm equally interested in seeing an authentic phylogenetic tree of Panaeolus, but I've been experimenting with both the NCBI Nucleotide BLAST site and the MAFFT Phylogenetic Tree site, and both are currently producing highly flawed and highly incomplete results.

Let's talk Psilocybe for a second. And to make to things more accurate, let's also discuss this within the ITS1 region.

When we compare a bunch of species we don't want to randomly compare genes or regions. That doesn't make sense and is completely devoid of reason (ie compare to the purpose of DNA barcoding).

The Advanced Search at the NCBI nucleotide site is not currently functioning at it should - and that should be obvious to the NCBI Helpdesk. However, if you simply type in "Psilocybe ITS1" at http://www.ncbi.nlm.nih.gov/nuccore and hit Search, it will come up currently with 75 search results.

Issue No. 1: Almost immediately you will notice that there are old species that were once in the genus Psilocybe and have been moved into the genus Deconica, but they are still listed on NBCI as being in the genus Psilocybe. Who wants to email them the genus/species lists for these to genera? Please, someone intervene.

Issue No. 2: As most of us know, there are well over 100 species, currently, listed in the genus Psilocybe. The NCBI database is missing most of these species' sequences. If Douglas Smith or anyone else has successfully constructed an ITS1 and/or ITS2 tree that is more complete and accurate please post it here.

Issue No. 3: The MAFFT phylogenetic tree that I produced based on the 75 Psilocybes produced a tree that does not coherently display ancestry. This is the whole point of a phylogenetic tree - to make sense of the Common Ancestor theory and to show which species mutated over time to form new species.

Please - all Hornets rise! All Hornest get angry and overcome this! Email these Helpdesks! Call your senators and representatives, your teachers, your students, you colleagues, your friends......


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OfflineAlan RockefellerM
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Re: Panaeolus phylogenetic tree [Re: The Lightning]
    #18166465 - 04/25/13 01:44 PM (10 years, 9 months ago)

Quote:

Issue No. 1: Almost immediately you will notice that there are old species that were once in the genus Psilocybe and have been moved into the genus Deconica, but they are still listed on NBCI as being in the genus Psilocybe. Who wants to email them the genus/species lists for these to genera? Please, someone intervene.




They don't update their DB when new names are published.  It's up to you to cut out the sequences which are not in Psilocybe.


Quote:

Issue No. 2: As most of us know, there are well over 100 species, currently, listed in the genus Psilocybe. The NCBI database is missing most of these species' sequences.





An effort should be made to gather and sequence species that are not yet in genbank.  Cyanofibrillosa is done, baeocystis should be done soon.  What else do we have access to that isn't in there?


Quote:

Issue No. 3: The MAFFT phylogenetic tree that I produced based on the 75 Psilocybes produced a tree that does not coherently display ancestry. This is the whole point of a phylogenetic tree - to make sense of the Common Ancestor theory and to show which species mutated over time to form new species.





How well are your sequences aligning?    You might need to toss out some sequences that don't have significant overlap.  And definitely toss out the Deconica sequences.


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Invisiblemaynardjameskeenan
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Re: Panaeolus phylogenetic tree [Re: Alan Rockefeller]
    #18166509 - 04/25/13 01:56 PM (10 years, 9 months ago)

Quote:

Alan Rockefeller said:
Quote:

Issue No. 1: Almost immediately you will notice that there are old species that were once in the genus Psilocybe and have been moved into the genus Deconica, but they are still listed on NBCI as being in the genus Psilocybe. Who wants to email them the genus/species lists for these to genera? Please, someone intervene.




They don't update their DB when new names are published.  It's up to you to cut out the sequences which are not in Psilocybe.


Quote:

Issue No. 2: As most of us know, there are well over 100 species, currently, listed in the genus Psilocybe. The NCBI database is missing most of these species' sequences.





An effort should be made to gather and sequence species that are not yet in genbank.  Cyanofibrillosa is done, baeocystis should be done soon.  What else do we have access to that isn't in there?


Quote:

Issue No. 3: The MAFFT phylogenetic tree that I produced based on the 75 Psilocybes produced a tree that does not coherently display ancestry. This is the whole point of a phylogenetic tree - to make sense of the Common Ancestor theory and to show which species mutated over time to form new species.





How well are your sequences aligning?    You might need to toss out some sequences that don't have significant overlap.  And definitely toss out the Deconica sequences.



Maybe someone (me) can make a list of the species that still need sequenced and make a thread to let other know what is needed...

Why don't they test all collections the same way? Ie.

-Psilocybe cyanescens voucher PRM 902040 18S small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S large subunit ribosomal RNA gene, partial sequence

-Psilocybe cyanescens voucher PRM 901481 translation elongation factor 1-alpha (EF1-alpha) gene, partial cds

I am correct in thinking these are tests in difference area of the genetic sequence?


--------------------
May you be filled with loving kindness.
May you be well.
May you be peaceful and at ease.
May you be happy.



AMU Q&A


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OfflineAlan RockefellerM
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Re: Panaeolus phylogenetic tree [Re: maynardjameskeenan]
    #18166563 - 04/25/13 02:09 PM (10 years, 9 months ago)

Quote:

maynardjameskeenan said:
Why don't they test all collections the same way? Ie.

-Psilocybe cyanescens voucher PRM 902040 18S small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S large subunit ribosomal RNA gene, partial sequence

-Psilocybe cyanescens voucher PRM 901481 translation elongation factor 1-alpha (EF1-alpha) gene, partial cds

I am correct in thinking these are tests in difference area of the genetic sequence?





Correct, those are two different loci and they can not be directly compared because there is no overlap.  ITS is often the best gene region for finding relationships between species.  LSU and ef-1 are better for determining the relationships between genera.


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Re: Panaeolus phylogenetic tree [Re: Alan Rockefeller]
    #18166713 - 04/25/13 02:37 PM (10 years, 9 months ago)

Quote:

What else do we have access to that isn't in there?




I'll repeat this to others because I'm sure there's important data out there: Who knows of some research that did not get into Genbank (NCBI)?

Quote:

How well are your sequences aligning?




I hate it when you make me ask you questions. I feel stupid when I ask questions. Can you tell us the difference between a sequence, a BLAST, and a sequence alignment? If you have an extra moment or two, can you also show us the difference between a poorly aligned sequence and a well-aligned sequence?

Quote:

Maybe someone (me) can make a list of the species that still need sequenced and make a thread to let other know what is needed.




Please do. I would benefit from it and it'll keep things rolling.


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Invisiblemaynardjameskeenan
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Re: Panaeolus phylogenetic tree [Re: The Lightning]
    #18166939 - 04/25/13 03:03 PM (10 years, 9 months ago)

Quote:

The Lightning said:


Quote:

How well are your sequences aligning?




I hate it when you make me ask you questions. I feel stupid when I ask questions. Can you tell us the difference between a sequence, a BLAST, and a sequence alignment? If you have an extra moment or two, can you also show us the difference between a poorly aligned sequence and a well-aligned sequence?






I think this is what he means.


aligned meaning that the same area of the sequence are compared to one another.
If the same regions of the sequence aren't compared it gives erroneous results.
They need to have overlapping sequences to be meaningful, it determines common ancestry I think if they share common areas of their sequences IE. same genes in the same places. 
It's like asking what's the make and model of your car and I say blue, unless certain aspects of the sequence are compared it doesn't mean much at all. I'm not certain to be honest, I am still kinda new to this.

Why the hell don't they test all samples the same way?

I know what you mean about Alan making you feel dumb, he does it to me too... :dunce:

Quote:

What else do we have access to that isn't in there?




Quote:

The Lightning said:
I'll repeat this to others because I'm sure there's important data out there: Who knows of some research that did not get into Genbank (NCBI)?




This is easy to find out, just search the Genbank for any given species and if it doesn't return any results that means it doesn't have an entry, Psilocybe strictipes is a good example.



Edited by maynardjameskeenan (04/25/13 03:13 PM)


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Invisiblemaynardjameskeenan
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Re: Panaeolus phylogenetic tree [Re: maynardjameskeenan]
    #18167102 - 04/25/13 03:29 PM (10 years, 9 months ago)

I think sometimes Alan is intentionally obtuse so I will find things out for myself,
It's probably more meaningful to find things out on your own then to just get the answers from somebody. 

I wish he would explain things to me like I'm 5 sometimes.


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May you be well.
May you be peaceful and at ease.
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InvisibleThe Lightning
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Re: Panaeolus phylogenetic tree [Re: maynardjameskeenan]
    #18167171 - 04/25/13 03:42 PM (10 years, 9 months ago)

Quote:

Why the hell don't they test all samples the same way?




DNA sequencing is an attempt at formal species identification. Different life forms (birds vs fungi for instance) can have different regions or genes that are more accurate, more legible, or more determinative - so for birds you might want to use CO1 and for fungi you might want to use ITS1.

There have been proposals and articles written as recently as the past two years about which area is most relevant for fungi.

http://www.pnas.org/content/early/2012/03/29/1117018109.full.pdf

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025081

http://www.springerprotocols.com/Pdf/doi/10.1007/978-1-61779-591-6_9?encCode=TkVHOjlfNi0xOTUtOTc3MTYtMS04Nzk=&tokenString=XXcLRlQOVJlxS+34hSUhsw==&access=denied

http://www.benthamscience.com/open/toainfoj/articles/V005/SI0001TOAINFOJ/45TOAINFOJ.pdf


Quote:

It's probably more meaningful to find things out on your own




Not always. Alan and others have really been helpful to me. I'm thankful for their answers.


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Invisiblemaynardjameskeenan
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Re: Panaeolus phylogenetic tree [Re: The Lightning]
    #18167321 - 04/25/13 04:07 PM (10 years, 9 months ago)

Quote:

The Lightning said:
Quote:

Why the hell don't they test all samples the same way?




DNA sequencing is an attempt at formal species identification. Different life forms (birds vs fungi for instance) can have different regions or genes that are more accurate, more legible, or more determinative - so for birds you might want to use CO1 and for fungi you might want to use ITS1.

There have been proposals and articles written as recently as the past two years about which area is most relevant for fungi.

http://www.pnas.org/content/early/2012/03/29/1117018109.full.pdf

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025081

http://www.springerprotocols.com/Pdf/doi/10.1007/978-1-61779-591-6_9?encCode=TkVHOjlfNi0xOTUtOTc3MTYtMS04Nzk=&tokenString=XXcLRlQOVJlxS+34hSUhsw==&access=denied

http://www.benthamscience.com/open/toainfoj/articles/V005/SI0001TOAINFOJ/45TOAINFOJ.pdf




Makes sense. Thank you.

Quote:

It's probably more meaningful to find things out on your own




Quote:

The Lightning said:
Not always. Alan and others have really been helpful to me. I'm thankful for their answers.




You make it sound as if I'm ungrateful...
I agree with you. I did say sometimes. He's been a great help to me over the past few years but at times he can seem intentionally vague, IMO.


--------------------
May you be filled with loving kindness.
May you be well.
May you be peaceful and at ease.
May you be happy.



AMU Q&A


Edited by maynardjameskeenan (04/25/13 04:10 PM)


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InvisibleThe Lightning
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Re: Panaeolus phylogenetic tree [Re: maynardjameskeenan]
    #18167548 - 04/25/13 04:54 PM (10 years, 9 months ago)

Quote:

You make it sound as if I'm ungrateful...




No, my friend. I didn't mean it that way at all. Sometimes my typed-tone doesn't translate the actual way I intended it to sound.


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Re: Panaeolus phylogenetic tree [Re: The Lightning]
    #18198462 - 05/01/13 06:15 PM (10 years, 8 months ago)

Quote:

Can you tell us the difference between a sequence, a BLAST, and a sequence alignment?




Sequence

A nucleic acid sequence is a succession of letters that indicate the order of nucleotides within a DNA (using GACT) or RNA (GACU) molecule.


Example of a sequence of Psilocybe ovoideocystidiata:

1 gatcattatt gaataacttt ggcgtggttg tagctggccc tctcgggggc atgtgctcgc
      61 cygtcatctt tatatctcca cctgtgcacc ttttgtagac gtctttgttg gaagctgrat
      121 aggagagaat gggtgctagt cactctttct cgagttgaag gctttctcaa ggtcgctcta
      181 tgttttcata taccccaagt atgtaacaga atgtatctat atggccttgt gcctataaaa
      241 ctatatacaa ctttcagcaa cggatctctt ggctctcgca tcgatgaaga acgcagcgaa
      301 atgcgataag taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacctt
      361 gcgctccttg gtattccgag gagcatgcct gtttgagtgt cattaaattc tcaaccttac
      421 cagcttttgt tagcttgtgt aatggcttgg acttgggggt tttttgccgg cttctaacaa
      481 agtcagctcc ccttaaatgc attagccggc tgcccgctgt ggaccgtcta ttggtgtgat
      541 aattatctac gccgtggatg tctgctatca atgggttttt aaagctgctt ctaaccgtct
      601 gttcattcgg acaatacaat gacaatttga cctcaaatca ggtaggacta cccgctgaac
      661 ttaagcatat ca


BLAST (Basic Local Alignment Search Tool)

BLAST finds regions of similarity between biological sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.


Sequence Alignment

A sequence alignment is a way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences. Aligned sequences of nucleotides are typically displayed in rows.



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Re: Panaeolus phylogenetic tree [Re: The Lightning]
    #18198536 - 05/01/13 06:32 PM (10 years, 8 months ago)

Quote:

The Lightning said:
Quote:

Can you tell us the difference between a sequence, a BLAST, and a sequence alignment?




Sequence

A nucleic acid sequence is a succession of letters that indicate the order of nucleotides within a DNA (using GACT) or RNA (GACU) molecule.


Example of a sequence of Psilocybe ovoideocystidiata:

1 gatcattatt gaataacttt ggcgtggttg tagctggccc tctcgggggc atgtgctcgc
      61 cygtcatctt tatatctcca cctgtgcacc ttttgtagac gtctttgttg gaagctgrat
      121 aggagagaat gggtgctagt cactctttct cgagttgaag gctttctcaa ggtcgctcta
      181 tgttttcata taccccaagt atgtaacaga atgtatctat atggccttgt gcctataaaa
      241 ctatatacaa ctttcagcaa cggatctctt ggctctcgca tcgatgaaga acgcagcgaa
      301 atgcgataag taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacctt
      361 gcgctccttg gtattccgag gagcatgcct gtttgagtgt cattaaattc tcaaccttac
      421 cagcttttgt tagcttgtgt aatggcttgg acttgggggt tttttgccgg cttctaacaa
      481 agtcagctcc ccttaaatgc attagccggc tgcccgctgt ggaccgtcta ttggtgtgat
      541 aattatctac gccgtggatg tctgctatca atgggttttt aaagctgctt ctaaccgtct
      601 gttcattcgg acaatacaat gacaatttga cctcaaatca ggtaggacta cccgctgaac
      661 ttaagcatat ca


BLAST (Basic Local Alignment Search Tool)

BLAST finds regions of similarity between biological sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.


Sequence Alignment

A sequence alignment is a way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences. Aligned sequences of nucleotides are typically displayed in rows.





:bow2: You are great at explaining things! Awesome, i wanna guide for DNA of mushrooms and using BLAST, ect. You should make me one for the guide :super:


--------------------
~~~~~~***Psilocybin Mushrooms***~~~~~~
_________A Practical Guide To Psilocybin Mushrooms_________

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"Mr. Joust, I see you don't actually partake in the psilocin, but it looks like it may partake in you!" -Gojira


       


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* I see blue! possible Copelandia cyanescens? mikonn 3,841 18 06/21/05 10:31 PM
by EonTan
* Auriscalpium Phylogenetic Tree Alan RockefellerM 3,199 7 08/10/11 02:21 PM
by Alan Rockefeller
* Copelandia Cyanescens!!!!!!!!!
( 1 2 3 4 ... 18 19 )
Beege 18,293 362 09/29/08 03:53 PM
by fishfarmin
* Copelandia Cyanescens CaseyHook 2,900 9 09/19/05 11:45 PM
by CptnGarden
* Copelandia Cyans - or just exact look-a-likes!? oO_wombat_Oo 1,816 8 03/28/02 06:55 PM
by mjshroomer
* Copelandia Cyanescens in New Zealand?
( 1 2 all )
montmont 20,514 31 06/08/15 03:24 AM
by Anglerfish
* Possible Copelandia? DarkSideMoon 1,273 6 08/19/06 08:21 PM
by DarkSideMoon
* copelandia tropicalis and cyanescens nelgything 1,230 3 11/02/05 03:21 PM
by nelgything

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