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Link to this post \| posted 13 Jun, 2018 19:22
cdshaffer	Great questions. Pecaan does not save the actual alignments, it just saves the hit table with the results you see. If you want alignments you have to either use NCBI web based blast or DNA Master. As for the listing in the notes, the issue is the structure of the non-redundant database that is the default database everyone searches with BLAST. This database is made much smaller and easier to search by combining all identical sequences into a single entry. Thus if there are three phage genes with exactly the same amino acid sequence and you search the non-redundant protein database you will get one hit which will have three names. In the web NCBI blast results you will just see the title to one sequence and the rest are kind of hidden but you will see a link with a tiny triangle and word like "see 2 more Title(s)". If you click the link you would see the full descriptions for all the other proteins. For PECAAN it was decided to explicitly list out all three in the Description column since any one of the three genes could have the relevant functional annotation and thus all three are also used to create the full notes for the same reason. in your case the two submitted proteins "3-oxoadipate CoA-transferase" and "CoA transferase subunit A" are identical so they collapse into one entry in the non-redundant database with two descriptions. Edited 13 Jun, 2018 19:28

Link to this post | posted 13 Jun, 2018 19:22

Great questions.

Pecaan does not save the actual alignments, it just saves the hit table with the results you see. If you want alignments you have to either use NCBI web based blast or DNA Master.

As for the listing in the notes, the issue is the structure of the non-redundant database that is the default database everyone searches with BLAST. This database is made much smaller and easier to search by combining all identical sequences into a single entry. Thus if there are three phage genes with exactly the same amino acid sequence and you search the non-redundant protein database you will get one hit which will have three names. In the web NCBI blast results you will just see the title to one sequence and the rest are kind of hidden but you will see a link with a tiny triangle and word like "see 2 more Title(s)". If you click the link you would see the full descriptions for all the other proteins.

For PECAAN it was decided to explicitly list out all three in the Description column since any one of the three genes could have the relevant functional annotation and thus all three are also used to create the full notes for the same reason.

in your case the two submitted proteins "3-oxoadipate CoA-transferase" and "CoA transferase subunit A" are identical so they collapse into one entry in the non-redundant database with two descriptions.

Edited 13 Jun, 2018 19:28

Posted in: PECAAN → Issues with BLAST results in PECAAN

Link to this post \| posted 05 Jun, 2018 19:51
cdshaffer	Many of the O cluster phage have a derivative of MPME1, its clearly not a MPME2 but is rather a truncation. See this alignment as an example: Alignment to BPs_58 to JangDynasty gp22 (but several O phage have identical sequence): `Query 1 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS 60 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS Sbjct 1 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS 60 Query 61 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDML———————VVFD-YDTSPNK 112 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTD+ +FD +D+ P Sbjct 61 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDIFGTTSSGPTIFDSFDSGPGG 120 Query 113 A 113 Sbjct 121 V 121` As per Cresawn et al PLoS ONE 10(3) these should be annotated MPME1 even though they are truncations.

Link to this post | posted 05 Jun, 2018 19:51

cdshaffer

Many of the O cluster phage have a derivative of MPME1, its clearly not a MPME2 but is rather a truncation. See this alignment as an example:

Alignment to BPs_58 to JangDynasty gp22 (but several O phage have identical sequence):

Query  1    MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS  60
            MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS
Sbjct  1    MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS  60

Query  61   TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDML———————VVFD-YDTSPNK  112
            TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTD+         +FD +D+ P
Sbjct  61   TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDIFGTTSSGPTIFDSFDSGPGG  120

Query  113  A  113

Sbjct  121  V  121

As per Cresawn et al PLoS ONE 10(3) these should be annotated MPME1 even though they are truncations.

Posted in: Cluster O Annotation Tips → MPME

Link to this post \| posted 05 Jun, 2018 14:46
cdshaffer	It appears many of the O cluster phage have a derivative of MPME1, its clearly not a MPME2 but much more than a single residue difference: Alignment to BPs_58 to JangDynasty gp22 (but several O phage have identical sequence): `Query 1 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS 60 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS Sbjct 1 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS 60 Query 61 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDML———————VVFD-YDTSPNK 112 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTD+ +FD +D+ P Sbjct 61 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDIFGTTSSGPTIFDSFDSGPGG 120 Query 113 A 113 Sbjct 121 V 121` This is identical for 99 a.a. then what looks like a spurious BLAST extension, should we also call this MPME1 or add new approved term to somehow indicate a derivative of MPME1 As a follow up, in Cresawn et al PLoS ONE 10(3) they are annotating the "truncated" version with the full MPME1 annotation. I am going to add this to the O specific annotations forum Edited 05 Jun, 2018 19:45

Link to this post | posted 05 Jun, 2018 14:46

cdshaffer

It appears many of the O cluster phage have a derivative of MPME1, its clearly not a MPME2 but much more than a single residue difference:

Alignment to BPs_58 to JangDynasty gp22 (but several O phage have identical sequence):

Query  1    MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS  60
            MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS
Sbjct  1    MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS  60

Query  61   TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDML———————VVFD-YDTSPNK  112
            TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTD+         +FD +D+ P
Sbjct  61   TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDIFGTTSSGPTIFDSFDSGPGG  120

Query  113  A  113

Sbjct  121  V  121

This is identical for 99 a.a. then what looks like a spurious BLAST extension, should we also call this MPME1 or add new approved term to somehow indicate a derivative of MPME1
As a follow up, in Cresawn et al PLoS ONE 10(3) they are annotating the "truncated" version with the full MPME1 annotation. I am going to add this to the O specific annotations forum

Edited 05 Jun, 2018 19:45

Posted in: Cluster F Annotation Tips → MPMEs--which one?

Link to this post \| posted 29 May, 2018 21:26
cdshaffer	I think this is mostly an issue with synonyms and specificity of functional names. A quick check of Kegg shows that there are three entries for "thymidylate synthases". K00560 thyA, TYMS; thymidylate synthase [EC:2.1.1.45] K03465 thyX, thy1; thymidylate synthase (FAD) [EC:2.1.1.148] K13998 DHFR-TS; dihydrofolate reductase / thymidylate synthase [EC:1.5.1.3 2.1.1.45] Notice how the first and the last share a common EC number (ie they can carry out the same reaction). If you dig deeper into these entries you will see that all the thymidylate synthases are methytransferases and all three use dihydrofolate as the source of the methyl group and convert dUMP to dTMP (i.e. they are both a dihydofolate reductase and a thymidylate synthase). If you look farther down on the HHPRED search you will also see entries like this that are only ever so slightly lower probabilities which is also a hint as to the nature of the issue: 1J3K_A Bifunctional dihydrofolate reductase (E.C.1.5.1.3)-thymidylate synthase; bifunctional, OXIDOREDUCTASE, TRANSFERASE; HET: NDP, UMP, WRA; 2.1A {Plasmodium falciparum} SCOP: c.71.1.1 As for annotation, the current approved term is "ThyX-like thymidylate synthase"; but I am not sure that should be used here. Based on my brief investigation it appears that ThyX uses FADH while ThyA/DHFR-TS use NADH as coenzymes (hence the different EC numbers). Since all your HHPRED hits are to the DHFR-TS proteins they are likely not really "ThyX type" but rather a "ThyA type". So then the proper term used here should maybe be "ThyA-like thymidylate synthase" or simply "thymidylate synthase"; but someone should really double check the enzymology before adding anything to the approved list. Edited 29 May, 2018 21:30

Link to this post | posted 29 May, 2018 21:26

cdshaffer

I think this is mostly an issue with synonyms and specificity of functional names. A quick check of Kegg shows that there are three entries for "thymidylate synthases".

K00560
thyA, TYMS; thymidylate synthase [EC:2.1.1.45]
K03465
thyX, thy1; thymidylate synthase (FAD) [EC:2.1.1.148]
K13998
DHFR-TS; dihydrofolate reductase / thymidylate synthase [EC:1.5.1.3 2.1.1.45]

Notice how the first and the last share a common EC number (ie they can carry out the same reaction). If you dig deeper into these entries you will see that all the thymidylate synthases are methytransferases and all three use dihydrofolate as the source of the methyl group and convert dUMP to dTMP (i.e. they are both a dihydofolate reductase and a thymidylate synthase). If you look farther down on the HHPRED search you will also see entries like this that are only ever so slightly lower probabilities which is also a hint as to the nature of the issue:

1J3K_A Bifunctional dihydrofolate reductase (E.C.1.5.1.3)-thymidylate synthase; bifunctional, OXIDOREDUCTASE, TRANSFERASE; HET: NDP, UMP, WRA; 2.1A {Plasmodium falciparum} SCOP: c.71.1.1

As for annotation, the current approved term is "ThyX-like thymidylate synthase"; but I am not sure that should be used here. Based on my brief investigation it appears that ThyX uses FADH while ThyA/DHFR-TS use NADH as coenzymes (hence the different EC numbers). Since all your HHPRED hits are to the DHFR-TS proteins they are likely not really "ThyX type" but rather a "ThyA type". So then the proper term used here should maybe be "ThyA-like thymidylate synthase" or simply "thymidylate synthase"; but someone should really double check the enzymology before adding anything to the approved list.

Edited 29 May, 2018 21:30

Posted in: Functional Annotation → thymidylate synthases versus dihydrofolate reductases

Link to this post \| posted 22 May, 2018 00:07
cdshaffer	That message is a warning not an error. It comes up because it is easy to get duplicate genes if you use the DNA Master tool to merge multiple DNA Master files. In this case however, you have two genes, a short form and a long form of the TAC with the same 5' end and this is exactly what you want. So it is OK to ignore the warning.

Posted in: Cluster C Annotation Tips → frameshift

Link to this post \| posted 15 May, 2018 14:21
cdshaffer	OK, I did a quick google search on that error. It appears to be a WINE error code not a DNA Master error code. So, I tried a search using DNA Master on my mac with WINE and I too got that same OLE 80040152 error. I suggest you repost your question in the Running DNA Master on a Mac using Wine thread there are a few people who post there regularly with lots of WINE experience that might have seen and solved this issue. Edited 15 May, 2018 14:39

Link to this post | posted 15 May, 2018 14:21

cdshaffer

OK, I did a quick google search on that error. It appears to be a WINE error code not a DNA Master error code. So, I tried a search using DNA Master on my mac with WINE and I too got that same OLE 80040152 error. I suggest you repost your question in the Running DNA Master on a Mac using Wine thread there are a few people who post there regularly with lots of WINE experience that might have seen and solved this issue.

Edited 15 May, 2018 14:39

Posted in: DNA Master → DNAMaster BLAST failure

Link to this post \| posted 14 May, 2018 19:03
cdshaffer	just tried on my Win7 machine with the April 2018 update and a single BLAST search, that worked and was able to get results. Since it is peak hours I did not try a BLAST all. Edited 14 May, 2018 20:11

Posted in: DNA Master → DNAMaster BLAST failure

Link to this post \| posted 10 May, 2018 21:40
cdshaffer	I just help a student yesterday install the WINE version on yer mac and after we installed, ran the updater and restarted DNA Master things just didn't look right. So I had the student check in the Help -> about menu. Sure enough, the auto-update had failed even though I saw her run the update and there were no error messages. We re-ran the auto-update again and it worked the second time. So I would recommend you double check to make sure the installed version is the one from April 2018.

Link to this post | posted 10 May, 2018 21:40

cdshaffer

I just help a student yesterday install the WINE version on yer mac and after we installed, ran the updater and restarted DNA Master things just didn't look right. So I had the student check in the Help -> about menu. Sure enough, the auto-update had failed even though I saw her run the update and there were no error messages. We re-ran the auto-update again and it worked the second time. So I would recommend you double check to make sure the installed version is the one from April 2018.

Posted in: DNA Master → DNA Master and RBS data

Link to this post \| posted 06 May, 2018 20:55
cdshaffer	The updated phamerator database has now been fully run though starterator and the results have been posted. You will now find your report available: http://phages.wustl.edu/starterator/Pham45669Report.pdf Edited 07 May, 2018 03:12

Posted in: Starterator → Pham not found in Starterator

Link to this post \| posted 26 Apr, 2018 20:33
cdshaffer	Just a note in case someone comes to this thread later, in cases where the pack/unpack protocol doesn't work. Before you start over from scratch to redo all your annotations in a new DNA Master file you can try to at least transfer the notes and annotations using the technique Welkin describes in this post: https://seaphages.org/forums/topic/4547/?page=1#post-6264 That technique has saved me many times.

Link to this post | posted 26 Apr, 2018 20:33

cdshaffer

Just a note in case someone comes to this thread later, in cases where the pack/unpack protocol doesn't work. Before you start over from scratch to redo all your annotations in a new DNA Master file you can try to at least transfer the notes and annotations using the technique Welkin describes in this post:

https://seaphages.org/forums/topic/4547/?page=1#post-6264

That technique has saved me many times.

Posted in: DNA Master → Saving Blast Results in DNA Master

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