SEA-PHAGES | All posts created by cdshaffer

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Link to this post \| posted 01 Mar, 2023 21:55
cdshaffer	Not working for me on web page either. I did not test command line, but the error log for my web run shows that the program was not able to initialize at the biolib cloud service. Unfortunately, the web page and the command line both submit to biolib so if biolib is down not much can be done until they fix the service. I cannot seem to find any way to get the status of the biolib cloud service so if anyone knows how to that, would love to hear it. There is a way to run the whole analysis locally but it requires access to specialized GPU hardware to do the analysis so we are really just stuck waiting for biolib service to be restored. Hopefully they are aware of this issue and are working on a solution, again can't find a status page anywhere, so hard to know.

Link to this post | posted 01 Mar, 2023 21:55

Not working for me on web page either. I did not test command line, but the error log for my web run shows that the program was not able to initialize at the biolib cloud service. Unfortunately, the web page and the command line both submit to biolib so if biolib is down not much can be done until they fix the service.

I cannot seem to find any way to get the status of the biolib cloud service so if anyone knows how to that, would love to hear it.

There is a way to run the whole analysis locally but it requires access to specialized GPU hardware to do the analysis so we are really just stuck waiting for biolib service to be restored.

Hopefully they are aware of this issue and are working on a solution, again can't find a status page anywhere, so hard to know.

Posted in: Functional Annotation → Deep TMHMM?

Link to this post \| posted 29 Jan, 2023 22:44
cdshaffer	Just an FYI, I have been trying to reach PECAAN all day today with no success. Also, this website is also reporting it is down. Emails and messages have been sent to Clair and Dex.

Posted in: PECAAN → PECAAN Down?

Link to this post \| posted 11 Jan, 2023 20:24
cdshaffer	when presented with this issue of specificity (i.e. should one annotate using the more specific term that includes the "MerR like" ) I like to first check out the PFam hits; others prefer the CD hit database, both are fine, I am just used to the Pfam dataset and I think the Hidden markov models that underlie PFam database (instead of the PSSM data in CDD) are more sensitive. You can add the Pfam database to an hhpred search, I usually don't add this database by default but I would go back and add it in a situation like this. In this case, your protein hits PF13411.9 ; MerR_1 ; MerR HTH family regulatory protein with 98% and 75% alignment. So in terms of assessing the quality of the hit I would say it is borderline (for PFam hits I do like nearly full length) but since this is just a specificity issue (I.e. there is plenty of evidence from your search that there is indeed an HTH in this protein) I would tell a student that, yes, this PFam hit is sufficient evidence to support the added "MerR like" term to the function. Note that some of the other proteins in your search do also mention the MerR like domain but you need to be careful and not take that as evidence at face value. Since HHPRED will happily report partial alignments, to use that evidence you would need to confirm that the MerR like HTH domain in the description is also in the region of the alignment. This can easily be done, it just usually takes a few clicks to dig into other databases and thus more time; whereas when looking for a good PFam hit, all the evidence you need is right there on the HHpred results page, so it is more of an expediency issue than a "quality of evidence" issue as to why I prefer using PFam. Edited 11 Jan, 2023 20:24

Link to this post | posted 11 Jan, 2023 20:24

cdshaffer

when presented with this issue of specificity (i.e. should one annotate using the more specific term that includes the "MerR like" ) I like to first check out the PFam hits; others prefer the CD hit database, both are fine, I am just used to the Pfam dataset and I think the Hidden markov models that underlie PFam database (instead of the PSSM data in CDD) are more sensitive.

You can add the Pfam database to an hhpred search, I usually don't add this database by default but I would go back and add it in a situation like this. In this case, your protein hits PF13411.9 ; MerR_1 ; MerR HTH family regulatory protein with 98% and 75% alignment. So in terms of assessing the quality of the hit I would say it is borderline (for PFam hits I do like nearly full length) but since this is just a specificity issue (I.e. there is plenty of evidence from your search that there is indeed an HTH in this protein) I would tell a student that, yes, this PFam hit is sufficient evidence to support the added "MerR like" term to the function.

Note that some of the other proteins in your search do also mention the MerR like domain but you need to be careful and not take that as evidence at face value. Since HHPRED will happily report partial alignments, to use that evidence you would need to confirm that the MerR like HTH domain in the description is also in the region of the alignment. This can easily be done, it just usually takes a few clicks to dig into other databases and thus more time; whereas when looking for a good PFam hit, all the evidence you need is right there on the HHpred results page, so it is more of an expediency issue than a "quality of evidence" issue as to why I prefer using PFam.

Edited 11 Jan, 2023 20:24

Posted in: Annotation → helix-turn-helix binding domain or protein?

Link to this post \| posted 19 Dec, 2022 23:20
cdshaffer	Ok I just tried this out and everything worked. I now have DNA Master running in a Windows 11 for ARM using the free "for personal use" version of VMWare Fusion 13 so this is a solution that does not require buying parallels. With all the registering, downloading and configuring it took about 2.5 hours. The video was very detailed and goes through all the steps. So if you are at all reticent I recommend watching the video linked about. If you want to just try I would add the following steps: step 4 add that after download you need to install homebrew on your mac, then use homebrew to install the qemu suite, then use qemu-img to convert the Windows 11 client download from a VHDX format to a VMDK format for use with VMWare. step 7. you will get stuck trying to get windows to start up as installation now requires an internet connection but you cannot install the network drivers and get internet until you isntall the VMtools which you cannot do until you start up Windows. So check out the video or search for on how to boot windows 11 without internet. Finally to get everything working once Windows fully boots, use Powershell in administrator to run the VMWare Tools from the virtual CD. I too have not been able to get file sharing or copy/paste set up. According to this page: https://communities.vmware.com/t5/VMware-Fusion-Discussions/Can-t-enable-the-shared-folders-with-Fusion-13-Player-free/td-p/2941943 and this page: https://communities.vmware.com/t5/VMware-Fusion-Discussions/Copy-Paste-file-sharing-between-Ventura-and-Windows-11-ARM-doesn/m-p/2940220 these features are not yet supported yet for M1 mac/Windows 11 for Arm. See suggested work arounds.

Link to this post | posted 19 Dec, 2022 23:20

cdshaffer

Ok I just tried this out and everything worked. I now have DNA Master running in a Windows 11 for ARM using the free "for personal use" version of VMWare Fusion 13 so this is a solution that does not require buying parallels.

With all the registering, downloading and configuring it took about 2.5 hours. The video was very detailed and goes through all the steps. So if you are at all reticent I recommend watching the video linked about. If you want to just try I would add the following steps:

step 4 add that after download you need to install homebrew on your mac, then use homebrew to install the qemu suite, then use qemu-img to convert the Windows 11 client download from a VHDX format to a VMDK format for use with VMWare.

step 7. you will get stuck trying to get windows to start up as installation now requires an internet connection but you cannot install the network drivers and get internet until you isntall the VMtools which you cannot do until you start up Windows. So check out the video or search for on how to boot windows 11 without internet.
Finally to get everything working once Windows fully boots, use Powershell in administrator to run the VMWare Tools from the virtual CD.

I too have not been able to get file sharing or copy/paste set up. According to this page:
https://communities.vmware.com/t5/VMware-Fusion-Discussions/Can-t-enable-the-shared-folders-with-Fusion-13-Player-free/td-p/2941943
and this page:
https://communities.vmware.com/t5/VMware-Fusion-Discussions/Copy-Paste-file-sharing-between-Ventura-and-Windows-11-ARM-doesn/m-p/2940220
these features are not yet supported yet for M1 mac/Windows 11 for Arm. See suggested work arounds.

Posted in: DNA Master → DNA Master on M1 Mac

Link to this post \| posted 29 Sep, 2022 15:37
cdshaffer	if it is asking for the user name that is: `root` for the password use: `phage`

Link to this post | posted 29 Sep, 2022 15:37

cdshaffer

if it is asking for the user name that is:

root

for the password use:

phage

Posted in: Bioinformatic Tools and Analyses → PhamNexus on SEA-VM

Link to this post \| posted 18 Aug, 2022 22:16
cdshaffer	That was very helpful. The HHPRED results will only be available for a short while and so I am posting the protein sequence of the example above so that anyone who wishes can redo the search. >CAG9959048.1 RecB-like helicase [Campylobacter jejuni] MSQFEPFLALEASAGSGKTFALSVRFVALILKGARINEILALTFTKKAANEMQKRIIETFLNLEKENKTS ECNELCKLLGKDKEELISLRDAKKEEFLRTELKISTFDAFFGKILRVFALNLGLSSDFTMSEERLDVREI FLKLLKKDELKDLAYYINLVDEKENFFSELEKFYENAYFQNRPKILNPTKAYINKAYSELRSYCLGLTHV KNYKNLCDNFKSEVLDLSVFMQSSFMTKFESTKYLQDLESTNLHFSAKRMELINALNTYAIELENYKIAN LMNLLNHYSEAKNIFHKDKNTLNFQDVSKKVYELITSEFKDMIYFRLDGFISHLLIDEFQDTSVIQYQIL RPLIAELVSGEGVKKNRTFFYVGDKKQSIYRFRKGKKELFDLLKQEFSQIKSDNLNTNYRSKELLVDFVN ETFKEKIKDYKEQFALESKKGGFVRIVESKEQKVKNQAQEIKEKTLETLFEQINFLRSKNISYDDICILC WKNSDADMVLDFLREQNIPAFTQSNVLLENKASVRLVLEYAKYCIFGDEFYLVFLKELLGFEPRKITLDF SKNAMENVLFLIKELKLDLNDIALIQFIEYAKTKENFLKLLFEPCALKIVSEQNMGISIMSVHKSKGLEF DHVILLDSLSKNNSNNEDIMLEYDINQGWQLHIKDKIRELTKEPIYTLFKENITRANYEDDINKLYVAFT RAKESLIIIKRNEESVNGNYPSYFKGGFLNIHSQERGFLESKEQILSVKKESIQTLQKFEKITLQEIQSE ERLDSKELYFGNAFHFFMQNLKLPKGENFQILTQRCKSKFRHFLDESDFEKLFKRIEILLKNIQFQNLIG DGKLLKEQALSFNGEIKQLDLLALKDEEAFIIDYKTGLAMQDKHKEQVGTYKIAISEILQKDKVRAFIVY CLENEIQILEI

Link to this post | posted 18 Aug, 2022 22:16

cdshaffer

That was very helpful. The HHPRED results will only be available for a short while and so I am posting the protein sequence of the example above so that anyone who wishes can redo the search.

>CAG9959048.1 RecB-like helicase [Campylobacter jejuni]
MSQFEPFLALEASAGSGKTFALSVRFVALILKGARINEILALTFTKKAANEMQKRIIETFLNLEKENKTS
ECNELCKLLGKDKEELISLRDAKKEEFLRTELKISTFDAFFGKILRVFALNLGLSSDFTMSEERLDVREI
FLKLLKKDELKDLAYYINLVDEKENFFSELEKFYENAYFQNRPKILNPTKAYINKAYSELRSYCLGLTHV
KNYKNLCDNFKSEVLDLSVFMQSSFMTKFESTKYLQDLESTNLHFSAKRMELINALNTYAIELENYKIAN
LMNLLNHYSEAKNIFHKDKNTLNFQDVSKKVYELITSEFKDMIYFRLDGFISHLLIDEFQDTSVIQYQIL
RPLIAELVSGEGVKKNRTFFYVGDKKQSIYRFRKGKKELFDLLKQEFSQIKSDNLNTNYRSKELLVDFVN
ETFKEKIKDYKEQFALESKKGGFVRIVESKEQKVKNQAQEIKEKTLETLFEQINFLRSKNISYDDICILC
WKNSDADMVLDFLREQNIPAFTQSNVLLENKASVRLVLEYAKYCIFGDEFYLVFLKELLGFEPRKITLDF
SKNAMENVLFLIKELKLDLNDIALIQFIEYAKTKENFLKLLFEPCALKIVSEQNMGISIMSVHKSKGLEF
DHVILLDSLSKNNSNNEDIMLEYDINQGWQLHIKDKIRELTKEPIYTLFKENITRANYEDDINKLYVAFT
RAKESLIIIKRNEESVNGNYPSYFKGGFLNIHSQERGFLESKEQILSVKKESIQTLQKFEKITLQEIQSE
ERLDSKELYFGNAFHFFMQNLKLPKGENFQILTQRCKSKFRHFLDESDFEKLFKRIEILLKNIQFQNLIG
DGKLLKEQALSFNGEIKQLDLLALKDEEAFIIDYKTGLAMQDKHKEQVGTYKIAISEILQKDKVRAFIVY
CLENEIQILEI

Posted in: Cluster P Annotation Tips → RecB-like exonuclease/helicase or Cas4 family exonuclease?

Link to this post \| posted 08 Aug, 2022 19:10
cdshaffer	q1: no assembler that I know of is aware of scientific standards about which strand should be the top strand and which should be the bottom strand. These standards are determined in a community-by-community way and so vary from one system to another. For example in eukaryotes we typically use the standard set by the cytologists and how they present whole chromosomes. Thus, in a very high quality assembly (where we probably have evidence for the locations of centromeres and telomeres) we will publish the sequence to match the typical cytological display. The phagesdb community has standards for determination of base 1 and strand. For all our phage deterination of Base 1 determination depends on the type of phage end structure, while strand is usually picked so the structural genes are top strand and near the beginning of the sequence. Dan posted some videos here with lots of help on this but you need to be able to look at the raw assembly to answer some of these questions. Finally, a lot of published sequences are actually sequences of prophage and base 1 and orientation are set by the location of the insertion site and the standard orientation for the host genome. [based on your gene matching I think this is the case for NC_015296.1] There are a collection of phage like this in the phamerator database where the order and orientation of the sequence has been changed from the genbank record to a different order and orientation so as to match (as best as possible) the typical order in the phamerator database, this makes drawing and interpreting the comparison maps at phamerator.org much easier. Q2: I would recommend you set your base 1 and strand using a similar stratagy, that is to say, pick the base 1 and orientation to make the subsequent steps of comparison as easy as possible. But that of course depends on what you're comparing your genome to. The good news here is that DNA master has a nice feature if you want to "roll" the genome around to set a different base 1 as well as the ability to switch to the complementary strand.

Link to this post | posted 08 Aug, 2022 19:10

cdshaffer

q1: no assembler that I know of is aware of scientific standards about which strand should be the top strand and which should be the bottom strand. These standards are determined in a community-by-community way and so vary from one system to another. For example in eukaryotes we typically use the standard set by the cytologists and how they present whole chromosomes. Thus, in a very high quality assembly (where we probably have evidence for the locations of centromeres and telomeres) we will publish the sequence to match the typical cytological display.

The phagesdb community has standards for determination of base 1 and strand. For all our phage deterination of Base 1 determination depends on the type of phage end structure, while strand is usually picked so the structural genes are top strand and near the beginning of the sequence. Dan posted some videos here with lots of help on this but you need to be able to look at the raw assembly to answer some of these questions.

Finally, a lot of published sequences are actually sequences of prophage and base 1 and orientation are set by the location of the insertion site and the standard orientation for the host genome. [based on your gene matching I think this is the case for NC_015296.1] There are a collection of phage like this in the phamerator database where the order and orientation of the sequence has been changed from the genbank record to a different order and orientation so as to match (as best as possible) the typical order in the phamerator database, this makes drawing and interpreting the comparison maps at phamerator.org much easier.

Q2: I would recommend you set your base 1 and strand using a similar stratagy, that is to say, pick the base 1 and orientation to make the subsequent steps of comparison as easy as possible. But that of course depends on what you're comparing your genome to. The good news here is that DNA master has a nice feature if you want to "roll" the genome around to set a different base 1 as well as the ability to switch to the complementary strand.

Posted in: Newbler → Getting Started with Phage Assembly

Link to this post \| posted 04 Aug, 2022 20:43
cdshaffer	We worked on many of the proteins in this pham. I don't think any student has worked on this protein in an in-depth investigation so I spent about 30 minutes poking at the literature and looking at a member of this pham we have been working on this summer pepperwood_draft_32 (stop: 14017). Looking broadly at the hhpred and blast hits, there is clearly a very wide ranging family of proteins all like those in this pham. Also, pepperwood_draft_32 may not be the best member to represent the group as it is a bit shorter than the others ( but its annotated with the longest ORF). In looking over the paper associated with the high matching crystal 2C2U, I can see that many of the proteins in this family have dual functions, DNA binding and Iron sequestration. Most of these proteins appear to be expressed in bacteria in responce to starvation or other forms of stress and are probably play a role in protecting DNA from damage during high levels of oxidative stress. The general name for proteins like this are DPS for DNA Protection during Starvation. In the paper on the 2C2U crystal they stated that the mechanism for DNA binding was not well understood, so i looked for recent papers and found this one: https://doi.org/10.1128/jb.00036-22 it appears to do a good job of summarizing these DPS proteins if you really want to get into the literature. But the one take home message I got from this is that proteins in this wide family have a variety of activities some do and some don't have DNA binding, some do and some don't store Iron, and some do and some don't have ferroxidase activity. So bottom line is that it is really going to be hard to get a good convincing argument of what function any particular protein has. So Bottom line I would say is that annotation in this case is about which kind of error you think is worse, false positives or false negatives. For functional annotation I prefer false negatives since too many people will take any annotation as "the truth". Many naive users may not understand that an annotation might mean something that is "weak but interesting". This propagation of incorrect functional annotations is a well understood problem with large databases like genbank and I would rather say nothing for the function and not exacerbate the problem. However, as you cited above, there is clearly some evidence to suggest a DNA binding activity for this protein and so it is totally reasonable to come to the conclusion that there is enough evidence to support this annotation. What we really have here is a situation where reasonable and well trained annotators can disagree and that is OK, without clear wet bench experimentation annotations are always just well informed estimations and it is totally reasonable to have different estimations.

Link to this post | posted 04 Aug, 2022 20:43

cdshaffer

We worked on many of the proteins in this pham. I don't think any student has worked on this protein in an in-depth investigation so I spent about 30 minutes poking at the literature and looking at a member of this pham we have been working on this summer pepperwood_draft_32 (stop: 14017). Looking broadly at the hhpred and blast hits, there is clearly a very wide ranging family of proteins all like those in this pham. Also, pepperwood_draft_32 may not be the best member to represent the group as it is a bit shorter than the others ( but its annotated with the longest ORF).

In looking over the paper associated with the high matching crystal 2C2U, I can see that many of the proteins in this family have dual functions, DNA binding and Iron sequestration. Most of these proteins appear to be expressed in bacteria in responce to starvation or other forms of stress and are probably play a role in protecting DNA from damage during high levels of oxidative stress. The general name for proteins like this are DPS for DNA Protection during Starvation.

In the paper on the 2C2U crystal they stated that the mechanism for DNA binding was not well understood, so i looked for recent papers and found this one: https://doi.org/10.1128/jb.00036-22

it appears to do a good job of summarizing these DPS proteins if you really want to get into the literature. But the one take home message I got from this is that proteins in this wide family have a variety of activities some do and some don't have DNA binding, some do and some don't store Iron, and some do and some don't have ferroxidase activity. So bottom line is that it is really going to be hard to get a good convincing argument of what function any particular protein has.

So Bottom line I would say is that annotation in this case is about which kind of error you think is worse, false positives or false negatives. For functional annotation I prefer false negatives since too many people will take any annotation as "the truth". Many naive users may not understand that an annotation might mean something that is "weak but interesting". This propagation of incorrect functional annotations is a well understood problem with large databases like genbank and I would rather say nothing for the function and not exacerbate the problem. However, as you cited above, there is clearly some evidence to suggest a DNA binding activity for this protein and so it is totally reasonable to come to the conclusion that there is enough evidence to support this annotation. What we really have here is a situation where reasonable and well trained annotators can disagree and that is OK, without clear wet bench experimentation annotations are always just well informed estimations and it is totally reasonable to have different estimations.

Posted in: Functional Annotation → DNA-binding ferritin-like protein

Link to this post \| posted 11 Jul, 2022 20:40
cdshaffer	To me the most informative piece of evidence is the start pattern in phage genes Kradal_87, Satis_87, EhyElimayoE_87. In the current version of the starterator report those are track 3. In that track you can see that start 4 is the longest orf (so I know those annotations cannot be too long) and that start 4 in Frankenweenie is 65470. So if Kradal, Satis and EhyElimayoE can make a perfectly viable phage with only the amino acids between start 4 and the stop codon, there is no reason to believe that the other phage require more amino acids. Yes this is a negative argument but lacking evidence to the contrary I will stick with parsimony as an important consideration. That is to say, in this case where there is not good evidence to the contrary, the simple conclusion that all the phage are using the same start (4) is the best annotation. So based mostly on parsimony, this pattern suggests to me that start 4 at 65470 should be annotated and not the start with the -4 overlap. Edited 11 Jul, 2022 20:44

Link to this post | posted 11 Jul, 2022 20:40

cdshaffer

To me the most informative piece of evidence is the start pattern in phage genes Kradal_87, Satis_87, EhyElimayoE_87. In the current version of the starterator report those are track 3. In that track you can see that start 4 is the longest orf (so I know those annotations cannot be too long) and that start 4 in Frankenweenie is 65470. So if Kradal, Satis and EhyElimayoE can make a perfectly viable phage with only the amino acids between start 4 and the stop codon, there is no reason to believe that the other phage require more amino acids. Yes this is a negative argument but lacking evidence to the contrary I will stick with parsimony as an important consideration. That is to say, in this case where there is not good evidence to the contrary, the simple conclusion that all the phage are using the same start (4) is the best annotation.

So based mostly on parsimony, this pattern suggests to me that start 4 at 65470 should be annotated and not the start with the -4 overlap.

Edited 11 Jul, 2022 20:44

Posted in: Cluster BM Annotation Tips → 4 bp overlap in Streptomyces phages?

Link to this post \| posted 17 Jun, 2022 19:07
cdshaffer	The upstream "G" form of this gene has a weak hit to the Tail assembly chaperone Pfam. . PF16459.8. The numbers on this hit are pretty bad 68% probability. So by itself it certainly not sufficient evidence to call a TAC. However we thought by adding in synteny evidence and that we had found a "pretty good" possible slippery sequence that the sum of all those observations that the TAC function was justified. But I agree with the recent discussions elsewhere that there should be a policy which is to only call the slip if there is a sequence match to one of the published, well documented slippery sequences. So calling the slip here is an overcall. If you remove the slip then there is really no evidence for functionally calling the downstream "T" region as a TAC (except for synteny evidence), and while we do call some gene functions based only on synteny, TAC is not one of them. So if we want to update the annotation in Genbank for this phage, I would say keep the TAC function call for the G form (32294-33103), {based on weak hhpred evidence combined with good synteny evidence), remove the gene that codes for the GT form (32294..33055,33057..33407), and add in a gene that starts at 33072 or 33126 as a gene, and give it an NKF annotation. I would probably go with the longer start without evidence to the contrary but since there is a very high level of uncertainty here in any case I could go with either start. 93Kb

Link to this post | posted 17 Jun, 2022 19:07

cdshaffer

The upstream "G" form of this gene has a weak hit to the Tail assembly chaperone Pfam. . PF16459.8. The numbers on this hit are pretty bad 68% probability. So by itself it certainly not sufficient evidence to call a TAC. However we thought by adding in synteny evidence and that we had found a "pretty good" possible slippery sequence that the sum of all those observations that the TAC function was justified. But I agree with the recent discussions elsewhere that there should be a policy which is to only call the slip if there is a sequence match to one of the published, well documented slippery sequences. So calling the slip here is an overcall.

If you remove the slip then there is really no evidence for functionally calling the downstream "T" region as a TAC (except for synteny evidence), and while we do call some gene functions based only on synteny, TAC is not one of them. So if we want to update the annotation in Genbank for this phage, I would say keep the TAC function call for the G form (32294-33103), {based on weak hhpred evidence combined with good synteny evidence), remove the gene that codes for the GT form (32294..33055,33057..33407), and add in a gene that starts at 33072 or 33126 as a gene, and give it an NKF annotation. I would probably go with the longer start without evidence to the contrary but since there is a very high level of uncertainty here in any case I could go with either start.

Posted in: Cluster BM Annotation Tips → Frameshift in BM cluster phages?

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