SEA-PHAGES | All posts created by cdshaffer

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Link to this post \| posted 16 May, 2020 18:39
cdshaffer	NCBI is quite restrictive in running BLAST searches that are submitted by computer programs like DNA Master and not submitted by individuals using the web interface. NCBI is really just not able to keep up with all the blast searches everyone around the world wants and so these automatically submitted searches are getting more and more restrictive. Your BLAST searches at NCBI do not stop if you quit DNA Master, so every time you retry the searches you add more and more searches to your queue and there is a limit to how many searches you can do at any one time. Given you have tried 7 times this is probably why you are getting this "Too many outstanding requests" error. According to the DNA Master settings it will submit 25 searches at a time so you may have submitted up to 175 searches if DNA master really did submit 25 searches each of those 7 times. According to the rules on this page: "We will move searches of users who submit more than 100 searches in a 24 hour period to a slower queue, or, in extreme cases, will block the requests. " Given that you used PECAAN all the blast results are available to any one who is checking your annotations, so i would suggest you go ahead and submit your DNA Master file without redoing the BLAST results and just note in the cover sheet the issues you had with NCBI and that the BLAST results are available in PECAAN.

Link to this post | posted 16 May, 2020 18:39

NCBI is quite restrictive in running BLAST searches that are submitted by computer programs like DNA Master and not submitted by individuals using the web interface. NCBI is really just not able to keep up with all the blast searches everyone around the world wants and so these automatically submitted searches are getting more and more restrictive. Your BLAST searches at NCBI do not stop if you quit DNA Master, so every time you retry the searches you add more and more searches to your queue and there is a limit to how many searches you can do at any one time. Given you have tried 7 times this is probably why you are getting this "Too many outstanding requests" error. According to the DNA Master settings it will submit 25 searches at a time so you may have submitted up to 175 searches if DNA master really did submit 25 searches each of those 7 times.

According to the rules on this page: "We will move searches of users who submit more than 100 searches in a 24 hour period to a slower queue, or, in extreme cases, will block the requests. "

Given that you used PECAAN all the blast results are available to any one who is checking your annotations, so i would suggest you go ahead and submit your DNA Master file without redoing the BLAST results and just note in the cover sheet the issues you had with NCBI and that the BLAST results are available in PECAAN.

Posted in: PECAAN → Problem BLASTING after PECAAN file transferred to DNA Master

Link to this post \| posted 27 Apr, 2020 00:44
cdshaffer	yes, two trivial things to try, 1) try a different browser sometimes this helps. 2) maximize the window size on the browser to fill the screen. 3) set zoom level to less than 100%, 4) Move to a computer with a large screen and try there (these problems usually show up on portables with small 12 or 13 inch screens). I never have had an issue in my office with my large 24 inch monitor

Posted in: PECAAN → problem with adding a gene

Link to this post \| posted 26 Mar, 2020 18:30
cdshaffer	missing or broken links are almost always due to pham changes with each new database release. New databases typically come out at least once a week. Once a database has been released by the Hatfull lab it takes each subsystem a variable amount of time to update. phagesdb.org will update almost immediately since it is also in the Hatfull lab. Phamerator.org and Pecaan will typically update pretty quickly as all they need to do is download and install the new database. Starterator will almost always update last as it takes quite a bit of time to run 15,000 clustal alignments and create the 15,000 reports. Phamerator.org, pecaan and starterator all have a way to see which database version they are on: phagesdb see: http://phamerator.webfactional.com/databases_Hatfull/Actino_Draft.version starterator see: http://phages.wustl.edu/starterator/database.version pecaan: look on any "pham maps" page just above the map phamerator.org: open the pull down menu in the top left

Link to this post | posted 26 Mar, 2020 18:30

cdshaffer

missing or broken links are almost always due to pham changes with each new database release.
New databases typically come out at least once a week.
Once a database has been released by the Hatfull lab it takes each subsystem a variable amount of time to update. phagesdb.org will update almost immediately since it is also in the Hatfull lab. Phamerator.org and Pecaan will typically update pretty quickly as all they need to do is download and install the new database. Starterator will almost always update last as it takes quite a bit of time to run 15,000 clustal alignments and create the 15,000 reports.

Phamerator.org, pecaan and starterator all have a way to see which database version they are on:
phagesdb see: http://phamerator.webfactional.com/databases_Hatfull/Actino_Draft.version
starterator see: http://phages.wustl.edu/starterator/database.version
pecaan: look on any "pham maps" page just above the map
phamerator.org: open the pull down menu in the top left

Posted in: PECAAN → Could this Pham be improperly assigned in PECAAN?

Link to this post \| posted 18 Mar, 2020 03:34
cdshaffer	This is a database synchronization issue. Phagesdb was on version 347 and the other sites were behind. This can happen anytime a new database is released. It takes a variable amount of time for each site to "catch up". Starterator usually takes about a day as there is a lot of computer work to calculate ~15000 multiple sequence alignments and generate ~15,000 reports, but it is now up to date and should give the correct results. Not sure about PECAAN or phamerator.org. The first thing you should try when you get these discrepancies is check which database each site is on.If they are on different versions This is how you can get the info on which version each site is on: phagesdb see: http://phamerator.webfactional.com/databases_Hatfull/Actino_Draft.version starterator see: http://phages.wustl.edu/starterator/database.version pecaan: look on any "pham maps" page just above the map phamerator.org: open the pull down menu in the top left

Link to this post | posted 18 Mar, 2020 03:34

cdshaffer

This is a database synchronization issue. Phagesdb was on version 347 and the other sites were behind. This can happen anytime a new database is released. It takes a variable amount of time for each site to "catch up". Starterator usually takes about a day as there is a lot of computer work to calculate ~15000 multiple sequence alignments and generate ~15,000 reports, but it is now up to date and should give the correct results. Not sure about PECAAN or phamerator.org. The first thing you should try when you get these discrepancies is check which database each site is on.If they are on different versions

This is how you can get the info on which version each site is on:
phagesdb see: http://phamerator.webfactional.com/databases_Hatfull/Actino_Draft.version
starterator see: http://phages.wustl.edu/starterator/database.version
pecaan: look on any "pham maps" page just above the map
phamerator.org: open the pull down menu in the top left

Posted in: Starterator → Phage not found in track

Link to this post \| posted 17 Mar, 2020 18:37
cdshaffer	I just searched the phamerator database. There are no genes from amy phage with host streptomyces that have lysin B as an annotation.

Posted in: Cluster BD Annotation Tips → lysin A

Link to this post \| posted 10 Mar, 2020 16:41
cdshaffer	There is a lot of help on the "data" page at phagesdb: Data page If you or someone you know has basic scripting skills you can use the API at phagesdb. It gives you the most flexibility and reproducibility if you want to repeat the experiment later. Failing that I would use the bulk system at NCBI. Here is a shorthand protocol I would use: 1. Get "Full tab delimited all phages spreadsheet" from the data page linked above. 2. Use your or a friend's excel skills to filter on cluster/subcluster to create a list with just the phage you want and select and copy the accession numbers column to clipboard 3. Go to Genbank and search the "nucleotide" database with the complete list of all the accession numbers (just paste the whole list in the little search box) 4. On the results page use the tiny menus near the top to change "Summary" to FASTA and then use the "Send to" menu and select "File". You should get a concatenated fasta file wherever your browser downloads. File will be named "sequence.fasta" which you should rename to something move appropriate like "A1_phages.fasta"

Link to this post | posted 10 Mar, 2020 16:41

cdshaffer

There is a lot of help on the "data" page at phagesdb: Data page

If you or someone you know has basic scripting skills you can use the API at phagesdb. It gives you the most flexibility and reproducibility if you want to repeat the experiment later.

Failing that I would use the bulk system at NCBI. Here is a shorthand protocol I would use:

1. Get "Full tab delimited all phages spreadsheet" from the data page linked above.
2. Use your or a friend's excel skills to filter on cluster/subcluster to create a list with just the phage you want and select and copy the accession numbers column to clipboard
3. Go to Genbank and search the "nucleotide" database with the complete list of all the accession numbers (just paste the whole list in the little search box)
4. On the results page use the tiny menus near the top to change "Summary" to FASTA and then use the "Send to" menu and select "File". You should get a concatenated fasta file wherever your browser downloads. File will be named "sequence.fasta" which you should rename to something move appropriate like "A1_phages.fasta"

Posted in: Phage Biology → Downloading cluster specific fasta files

Link to this post \| posted 09 Mar, 2020 16:28
cdshaffer	OK the rule that "you must have a B, in order to have an A" makes sense to me. Thanks for the clarification.

Posted in: Cluster BD Annotation Tips → lysin A

Link to this post \| posted 09 Mar, 2020 00:38
cdshaffer	Based on this posting from Feb 2020: https://seaphages.org/forums/topic/5000/ Which I will repost here: `Just looked at 2 cluster AO2 phages, JKerns and StevieBAY. StevieBAY's gene 28 is an endolysin. It possesses all of domains we expect to see in an endolysin. Gene 64 codes for the CHAP endopeptidase domain of LysK (a Staphylococcus phage lysin gene). It is reported out as an endolysin also. We won't call either of them lysin A (Reserved only for mycobacteriophages for now, unless the phage genomes contains a lysin A and a lysin B.` And this in the approved terms (as of Feb 2020): `some arthrobacter and streptomyces phages have a single endolysin with domains not found in the Mycobacteriophages (like the CHAP domain). use "endolysin" rather than lysin A if the phage does not infect Mycobacterium and no lysin b can be identified.` The BD6 phage appear to have a lysin which is a very high quality HHpred hit (99.8 Prob, 97.2 coverage) to the N-ACETYLMURAMOYL-L-ALANINE AMIDASE crystal (2WKX_A) should annotation be the approved term "endolysin" or "lysin A, N-acetylmuramoyl-L-alanine amidase domain" Edited 09 Mar, 2020 00:50

Link to this post | posted 09 Mar, 2020 00:38

cdshaffer

Based on this posting from Feb 2020:
https://seaphages.org/forums/topic/5000/

Which I will repost here:

Just looked at 2 cluster AO2 phages, JKerns and StevieBAY. StevieBAY's
gene 28 is an endolysin. It possesses all of domains we expect to see in an
endolysin. Gene 64 codes for the CHAP endopeptidase domain of LysK (a
Staphylococcus phage lysin gene). It is reported out as an endolysin also.
We won't call either of them lysin A (Reserved only for mycobacteriophages
for now, unless the phage genomes contains a lysin A and a lysin B.

And this in the approved terms (as of Feb 2020):

some arthrobacter and streptomyces phages have a single endolysin
with domains not found in the Mycobacteriophages (like the CHAP domain).
use "endolysin" rather than lysin A if the phage does not infect
Mycobacterium and no lysin b can be identified.

The BD6 phage appear to have a lysin which is a very high quality HHpred hit (99.8 Prob, 97.2 coverage) to the N-ACETYLMURAMOYL-L-ALANINE AMIDASE crystal (2WKX_A)

should annotation be the approved term "endolysin" or "lysin A, N-acetylmuramoyl-L-alanine amidase domain"

Edited 09 Mar, 2020 00:50

Posted in: Cluster BD Annotation Tips → lysin A

Link to this post \| posted 12 Feb, 2020 23:30
cdshaffer	Just a reminder to everyone that the most recent updates to Starterator were designed to improve the whole phage reports. These whole phage reports steamline some of the output that is needed for a whole pham report but is irrelevant if all you care about is annotating one particular phage. Unfortunately I cannot pre-compute whole starterator reports, its just too computationally intensive, but I am happy to generate a whole phage report for anyone by request. Simply post a follow-up message to this post or send me an email. All I need is the name of the phage and I can generate a report based on the initial auto-annotation. Be aware that for phage in large clusters it can take several hours of computation so expect some delay between request and response but it only takes me about 45 seconds to start the run and another 75 seconds to post the results so don't feel like this is a huge ask, happy to help. Also, you may find it helpful to run a whole phage report on a phage after you have worked on annotation and want to rerun the whole phage report based on those newer annotations. I can do that easily as well. Simply email me either your DNA Master file, or if you are using PECAAN export and send me the "CDS function" report and I can run a whole phage starterator report based on that new annotation set. Edited 13 Feb, 2020 03:26

Link to this post | posted 12 Feb, 2020 23:30

cdshaffer

Just a reminder to everyone that the most recent updates to Starterator were designed to improve the whole phage reports. These whole phage reports steamline some of the output that is needed for a whole pham report but is irrelevant if all you care about is annotating one particular phage. Unfortunately I cannot pre-compute whole starterator reports, its just too computationally intensive, but I am happy to generate a whole phage report for anyone by request. Simply post a follow-up message to this post or send me an email. All I need is the name of the phage and I can generate a report based on the initial auto-annotation. Be aware that for phage in large clusters it can take several hours of computation so expect some delay between request and response but it only takes me about 45 seconds to start the run and another 75 seconds to post the results so don't feel like this is a huge ask, happy to help.

Also, you may find it helpful to run a whole phage report on a phage after you have worked on annotation and want to rerun the whole phage report based on those newer annotations. I can do that easily as well. Simply email me either your DNA Master file, or if you are using PECAAN export and send me the "CDS function" report and I can run a whole phage starterator report based on that new annotation set.

Edited 13 Feb, 2020 03:26

Posted in: Starterator → whole phage starterator reports

Link to this post \| posted 08 Feb, 2020 00:29
cdshaffer	Yes there was a new version of the database posted this morning. Since each site updates as it can things can get out of sync. Not sure how you generated what you are calling your "gene list" but pecaan and phagesdb were updated earlier today so the links out of pecaan and out of phagesdb were pointing to pages that did not exist until starterator was also updated to the new version 339. This is how you can get the info on which version each site is on (links will only work between two sites if they are on the same version): phagesdb see: http://phamerator.webfactional.com/databases_Hatfull/Actino_Draft.version starterator see: http://phages.wustl.edu/starterator/database.version pecaan: look on any "pham maps" page just above the map phamerator.org: open the pull down menu in the top left Edited 08 Feb, 2020 00:30

Link to this post | posted 08 Feb, 2020 00:29

cdshaffer

Yes there was a new version of the database posted this morning. Since each site updates as it can things can get out of sync.
Not sure how you generated what you are calling your "gene list" but pecaan and phagesdb were updated earlier today so the links out of pecaan and out of phagesdb were pointing to pages that did not exist until starterator was also updated to the new version 339. This is how you can get the info on which version each site is on (links will only work between two sites if they are on the same version):
phagesdb see: http://phamerator.webfactional.com/databases_Hatfull/Actino_Draft.version
starterator see: http://phages.wustl.edu/starterator/database.version
pecaan: look on any "pham maps" page just above the map
phamerator.org: open the pull down menu in the top left

Edited 08 Feb, 2020 00:30

Posted in: Starterator → Pham not found in Starterator

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