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Link to this post \| posted 27 Mar, 2018 15:23
GregFrederick@letu.edu	We are using the accession numbers that we previously recorded from HHPred to reconfirm. But that too is taking a lot of extra time.

Posted in: DNA Master → HHPred Question - Concern

Link to this post \| posted 27 Mar, 2018 15:19
GregFrederick@letu.edu	There does not appear to be a forum section for HHPred topics. So I am placing this here for now. QUESTION-CONCERN: When we began the process this semester there were two SCOP databases available. Currently, there is only one SCOP database (e70_207) available. The other is no longer available in the selection list. So as we are reconfirming all our HHPred data notes, we are finding that the data found originally is no longer available. (Unfortunately, we are apparently also outside the 30 day window where HHPred retains the analysis. Therefore none of the previous query results are available on the website either.) Last year we had students saving each HHPred report as a PDF file on a shared drive. We decided to not do this in the current semester because students complained that it was too time consuming. But now since we no longer have access to the database, we cannot reconfirm those functions ID'd through the original HHPred analysis. QUESTION: HOW DO YOU ALL ARCHIVE ONLINE ANALYSES SUCH AS HHPred? DO YOU? Thanks in advance. Greg Edited 27 Mar, 2018 15:21

Link to this post | posted 27 Mar, 2018 15:19

GregFrederick@letu.edu

There does not appear to be a forum section for HHPred topics. So I am placing this here for now.

QUESTION-CONCERN: When we began the process this semester there were two SCOP databases available. Currently, there is only one SCOP database (e70_207) available. The other is no longer available in the selection list.

So as we are reconfirming all our HHPred data notes, we are finding that the data found originally is no longer available. (Unfortunately, we are apparently also outside the 30 day window where HHPred retains the analysis. Therefore none of the previous query results are available on the website either.)

Last year we had students saving each HHPred report as a PDF file on a shared drive. We decided to not do this in the current semester because students complained that it was too time consuming. But now since we no longer have access to the database, we cannot reconfirm those functions ID'd through the original HHPred analysis.

QUESTION: HOW DO YOU ALL ARCHIVE ONLINE ANALYSES SUCH AS HHPred? DO YOU?

Thanks in advance. Greg

Edited 27 Mar, 2018 15:21

Posted in: DNA Master → HHPred Question - Concern

Link to this post \| posted 06 Mar, 2018 16:27
GregFrederick@letu.edu	GregFrederick@letu.edu We really need help with this question. We have just about everything else completed. But this line: SIF-HHPred: For HHPred the user guide gives the following notes format. SIF-HHPred [NKF / function, database, phage name, gene number, database accession number, %alignment, probability] But HHPred (with the databases indicated in the User Manual) rarely finds phages. It does find conserved domains (and thus some possible functions). QUESTION:* I thought the purpose of HHPred was to attempt to ID previously undertermined functions or domains. Is this still correct? In the case of HHPred and following the instructions in the new User Guide we are looking for functions or conserved domains with a P value of 90% or greater. So based on the SIF-HHPred datascript line in the user guide and above, do we indicate "NKF" if we do not hit a phage. We have been listing identified conserved domains as we have in previous years. But the information required by the above datascript does not really fit that well. HELP??? . . Related question: The script for SIF-HHPred also asks for "%alignment" But the HHPRed website only give the following data for "hits". Probability: E-value: Score: Aligned Cols: Identities: Similarity: Is the percent alignment something we need to calculate? Or should we use one of the above provided values? If we need to calculate the percent alignment, can you please tell us how this should be done? Again, we are typically only finding small domains of 20 to 100 amino acid residues with this HHPred analysis so percent alignment values will normally be very very small if I understand correctly what you want. But please show us how to calculate these. Thank you. gf Edited 06 Mar, 2018 16:29

Link to this post | posted 06 Mar, 2018 16:27

GregFrederick@letu.edu

GregFrederick@letu.edu
We really need help with this question. We have just about everything else completed. But this line:

SIF-HHPred:

For HHPred the user guide gives the following notes format.

SIF-HHPred [NKF / function, database, phage name, gene number, database accession number, %alignment*, probability]

But HHPred (with the databases indicated in the User Manual) rarely finds phages. It does find conserved domains (and thus some possible functions).

QUESTION: I thought the purpose of HHPred was to attempt to ID previously undertermined functions or domains. Is this still correct?

In the case of HHPred and following the instructions in the new User Guide we are looking for functions or conserved domains with a P value of 90% or greater.

So based on the SIF-HHPred datascript line in the user guide and above, do we indicate "NKF" if we do not hit a phage. We have been listing identified conserved domains as we have in previous years. But the information required by the above datascript does not really fit that well.

HELP???

.
.
Related question: The script for SIF-HHPred also asks for "%alignment" But the HHPRed website only give the following data for "hits".

Probability: E-value: Score: Aligned Cols: Identities: Similarity:

Is the percent alignment something we need to calculate? Or should we use one of the above provided values?

If we need to calculate the percent alignment, can you please tell us how this should be done?

Again, we are typically only finding small domains of 20 to 100 amino acid residues with this HHPred analysis so percent alignment values will normally be very very small if I understand correctly what you want. But please show us how to calculate these. Thank you. gf

Edited 06 Mar, 2018 16:29

Posted in: Notes and Final Files → SIF-Blast; SIF-HHPred; SIF-Syn

Link to this post \| posted 06 Mar, 2018 16:09
GregFrederick@letu.edu	We really need help with this question. We have just about everything else completed. But this line: SIF-HHPred: For HHPred the user guide gives the following notes format. SIF-HHPred [NKF / function, database, phage name, gene number, database accession number, %alignment, probability] But HHPred (with the databases indicated in the User Manual) rarely finds phages. It does find conserved domains (and thus some possible functions). QUESTION:* I thought the purpose of HHPred was to attempt to ID previously undertermined functions or domains. Is this still correct? In the case of HHPred and following the instructions in the new User Guide we are looking for functions or conserved domains with a P value of 90% or greater. So based on the SIF-HHPred datascript line in the user guide and above, do we indicate "NKF" if we do not hit a phage. We have been listing identified conserved domains as we have in previous years. But the information required by the above datascript does not really fit that well. HELP???

Link to this post | posted 06 Mar, 2018 16:09

GregFrederick@letu.edu

We really need help with this question. We have just about everything else completed. But this line:

SIF-HHPred:

For HHPred the user guide gives the following notes format.

SIF-HHPred [NKF / function, database, phage name, gene number, database accession number, %alignment*, probability]

But HHPred (with the databases indicated in the User Manual) rarely finds phages. It does find conserved domains (and thus some possible functions).

QUESTION: I thought the purpose of HHPred was to attempt to ID previously undertermined functions or domains. Is this still correct?

In the case of HHPred and following the instructions in the new User Guide we are looking for functions or conserved domains with a P value of 90% or greater.

So based on the SIF-HHPred datascript line in the user guide and above, do we indicate "NKF" if we do not hit a phage. We have been listing identified conserved domains as we have in previous years. But the information required by the above datascript does not really fit that well.

HELP???

Posted in: Notes and Final Files → SIF-Blast; SIF-HHPred; SIF-Syn

Link to this post \| posted 22 Feb, 2018 17:03
GregFrederick@letu.edu	Wilken. I greatly appreciate you and your responses. We are trying to be diligent in providing accurate and valid information. So I have a few additional questions concerning the instructions in the new online guide related to the SIF reports. SIF-Blast: The instruction guide states to consider anything of e-4 or smaller. The manual states to report as follows: SIF-BLAST [NKF / function, database, phage name, gene number, database gene accession number, %alignment, evalue] We are using the NCBI website blast for this analysis (in addition to PhagesDB). Obviously, if we only hit hypothetical proteins, we enter "SIF-BLAST NKF" But if we see something that looks like a function in the NCBI Blast we report as follows (for example): function: as listed in NCBI database: NCBI (For this SIF-BLAST we are only using NCBI and not phagesDB. Is this the correct approach?) phage name: If the hit is a phage we list it. If it not a phage, do you want us to list the organism name??? gene number: This is not easy to find in the NCBI data. I think we may have found a way to get it. But the process is cumbersome. Do you need this gp# if the hit is not a phage. database gene accession number: This we can get through NCBI but not phagesDB. There is really no clarification in this section of the User Manual which BLAST source should be reported here. But since PhagesDB does not provide this number we are using NCBI. Is this correct? %alignment: NCBI provides this. Again, PhagesDB does not provide this value. Should this be corrected on the PhagesDB Blast result output? e value: We are looking for e-4 and smaller (closer to zero) and only reporting those. This is correct, right? I thought I remembered an e-value of e-16 and smaller last year. SIF-HHPred: For HHPred the user guide gives the following notes format. SIF-HHPred [NKF / function, database, phage name, gene number, database accession number, %alignment, probability] But HHPred (with the databases indicated in the User Manual) rarely finds phages. It does find conserved domains (and thus some possible functions). QUESTION:* I thought the purpose of HHPred was to attempt to ID previously undertermined functions or domains. Is this still correct? In the case of HHPred and following the instructions in the new User Guide we are looking for functions or conserved domains with a P value of 90% or greater. QUESTION: Do you still want us to list non-phage related functions with P-value of 90% or greater? If so, what is the correct syntax? SIF-SYNTENY: This note code is also new for us this year. So I want to make certain we are doing this correctly. We are looking at Synteny via Phamerator comparisons. Normally we pull up 3-5 phages in the same sub-cluster for comparison. QUESTION: Is comparing 3-5 members of the same cluster for synteny enough data? The code listed in the current version of the online User Guide lists the following code for SIF-SYNTENY. SIF-Syn: [NKF / function, phage(s) used to infer ] QUESTIONS: The code states "phage(s)" If there is only one, that is easy. But that raises the following questions. Q: According to the User Guide synteny can only be defined for the following 12 genes. Terminase Portal protein Capsid maturation protease Scaffolding protein Major capsid protein Major tail protein Tail assembly chaperones Tape measure protein Minor tail proteins lysin A holin lysin B But the notes options provided limit us to NKF or phage(s) list. NKF does not seem appropriate for those genes were we can actually see functions assigned in related phages. Q: In the case described directly above, is NKF correct or would NA more applicable? Q: If only 1 phage of 4-5 list a function, we list that function and phage. Do you also want to know that there are 37 (hypothetical number) phages that do not list that same function? Q: What if multiple functions are listed in multiple phages and multiple functions for a given feature, all of which are on the approved function name list as "approved". How do we list those? Do we list one or all phage/functions? We have found a few features that have approved function names (in one case three functions) with all of them on the approved list. We are going with the most specific function. But I am not certain that is y'alls desire. Q: How many surrounding genes should be considered in the assessment of Synteny? If there are any minimal limits on numbers of conserved features before we list a phage as SIF-SYN info? i.e. if two genes are adjacent in another solo phage in the same order as ours, with listed function, is that enough data to consider valuable from the synteny perspective? Lots of questions. Thank you in advance for taking time to help with these answers. Your time is much appreciated. Thank you. Edited 22 Feb, 2018 22:14

Link to this post | posted 22 Feb, 2018 17:03

GregFrederick@letu.edu

Wilken.

I greatly appreciate you and your responses. We are trying to be diligent in providing accurate and valid information. So I have a few additional questions concerning the instructions in the new online guide related to the SIF reports.

SIF-Blast:

The instruction guide states to consider anything of e-4 or smaller.

The manual states to report as follows:

SIF-BLAST [NKF / function, database, phage name, gene number, database gene accession number, %alignment, evalue]

We are using the NCBI website blast for this analysis (in addition to PhagesDB).

Obviously, if we only hit hypothetical proteins, we enter "SIF-BLAST NKF"

But if we see something that looks like a function in the NCBI Blast we report as follows (for example):

function: as listed in NCBI
database: NCBI (For this SIF-BLAST we are only using NCBI and not phagesDB. Is this the correct approach?)
phage name: If the hit is a phage we list it. If it not a phage, do you want us to list the organism name???
gene number: This is not easy to find in the NCBI data. I think we may have found a way to get it. But the process is cumbersome. Do you need this gp# if the hit is not a phage.
database gene accession number: This we can get through NCBI but not phagesDB. There is really no clarification in this section of the User Manual which BLAST source should be reported here. But since PhagesDB does not provide this number we are using NCBI. Is this correct?
%alignment: NCBI provides this. Again, PhagesDB does not provide this value. Should this be corrected on the PhagesDB Blast result output?
e value: We are looking for e-4 and smaller (closer to zero) and only reporting those. This is correct, right? I thought I remembered an e-value of e-16 and smaller last year.
SIF-HHPred:

For HHPred the user guide gives the following notes format.

SIF-HHPred [NKF / function, database, phage name, gene number, database accession number, %alignment*, probability]

But HHPred (with the databases indicated in the User Manual) rarely finds phages. It does find conserved domains (and thus some possible functions).

QUESTION: I thought the purpose of HHPred was to attempt to ID previously undertermined functions or domains. Is this still correct?

In the case of HHPred and following the instructions in the new User Guide we are looking for functions or conserved domains with a P value of 90% or greater.

QUESTION: Do you still want us to list non-phage related functions with P-value of 90% or greater? If so, what is the correct syntax?
SIF-SYNTENY:

This note code is also new for us this year. So I want to make certain we are doing this correctly.

We are looking at Synteny via Phamerator comparisons. Normally we pull up 3-5 phages in the same sub-cluster for comparison.

QUESTION: Is comparing 3-5 members of the same cluster for synteny enough data?

The code listed in the current version of the online User Guide lists the following code for SIF-SYNTENY.

SIF-Syn: [NKF / function, phage(s) used to infer ]

QUESTIONS: The code states "phage(s)" If there is only one, that is easy. But that raises the following questions.

Q: According to the User Guide synteny can only be defined for the following 12 genes.

Terminase
Portal protein
Capsid maturation protease
Scaffolding protein
Major capsid protein
Major tail protein
Tail assembly chaperones
Tape measure protein
Minor tail proteins
lysin A
holin
lysin B

But the notes options provided limit us to NKF or phage(s) list. NKF does not seem appropriate for those genes were we can actually see functions assigned in related phages.

Q: In the case described directly above, is NKF correct or would NA more applicable?

Q: If only 1 phage of 4-5 list a function, we list that function and phage. Do you also want to know that there are 37 (hypothetical number) phages that do not list that same function?

Q: What if multiple functions are listed in multiple phages and multiple functions for a given feature, all of which are on the approved function name list as "approved". How do we list those? Do we list one or all phage/functions? We have found a few features that have approved function names (in one case three functions) with all of them on the approved list. We are going with the most specific function. But I am not certain that is y'alls desire.

Q: How many surrounding genes should be considered in the assessment of Synteny?
If there are any minimal limits on numbers of conserved features before we list a phage as SIF-SYN info? i.e. if two genes are adjacent in another solo phage in the same order as ours, with listed function, is that enough data to consider valuable from the synteny perspective?

Lots of questions. Thank you in advance for taking time to help with these answers. Your time is much appreciated. Thank you.

Edited 22 Feb, 2018 22:14

Posted in: Notes and Final Files → SIF-Blast; SIF-HHPred; SIF-Syn

Link to this post \| posted 21 Feb, 2018 16:36
GregFrederick@letu.edu	Thank you. GF

Posted in: Notes and Final Files → SIF-Blast; SIF-HHPred; SIF-Syn

Link to this post \| posted 21 Feb, 2018 16:13
GregFrederick@letu.edu	One more clarification question Welkin: When you say "look in GenBank" you mean BLASTP against the NCBI database, correct?

Posted in: Notes and Final Files → SIF-Blast; SIF-HHPred; SIF-Syn

Link to this post \| posted 21 Feb, 2018 16:10
GregFrederick@letu.edu	Welkin Pope Hi Greg, Unfortunately, these data points are not redundant. The problem with assigning functions via only one source is that if an error has been introduced into the database, the error will be propagated in your annotation. The only way to make sure is to confirm your functional assignments through all sources. We are working to make all of the phage-related functional assigns match the entries in GEnBank— should happen shortly, in which case you would only need to look at one of these databases for functions in our phage genes. You should still look in GenBank for functions from sources outside of our database. And it is OK to relinquish genomes if you don't think you can do them all. I'd rather have fewer annotations done at a high standard than more that we have to fix on the back end. Best, Welkin Ok. Thanks. So if we use PECAAN (as we are for one larger genome) I understand that it does not currently include the SIF codes in the output. Does this mean that we need to manually go back in and enter them after we import from PECAAN? Thoughts?

Link to this post | posted 21 Feb, 2018 16:10

GregFrederick@letu.edu

Welkin Pope
Hi Greg,
Unfortunately, these data points are not redundant. The problem with assigning functions via only one source is that if an error has been introduced into the database, the error will be propagated in your annotation. The only way to make sure is to confirm your functional assignments through all sources. We are working to make all of the phage-related functional assigns match the entries in GEnBank— should happen shortly, in which case you would only need to look at one of these databases for functions in our phage genes. You should still look in GenBank for functions from sources outside of our database.

And it is OK to relinquish genomes if you don't think you can do them all. I'd rather have fewer annotations done at a high standard than more that we have to fix on the back end.

Best,
Welkin

Ok. Thanks. So if we use PECAAN (as we are for one larger genome) I understand that it does not currently include the SIF codes in the output.

Does this mean that we need to manually go back in and enter them after we import from PECAAN? Thoughts?

Posted in: Notes and Final Files → SIF-Blast; SIF-HHPred; SIF-Syn

Link to this post \| posted 20 Feb, 2018 22:32
GregFrederick@letu.edu	I just figured out what was going on with only auto-annotating the tRNA genes. I was working on a computer lab computer which I had set preferences on previously. But apparently DNA Master over-rode those preferences on an update at some point. Something reset the preferences to the default "Use NCBI servers". Anyway, once I went through again and reset the server settings as listed in the page below everything worked again. https://seaphages.org/media/docs/DNA_Master_Gene_Prediction_Settings.docx I hope this helps save headaches for someone else. Greg

Link to this post | posted 20 Feb, 2018 22:32

GregFrederick@letu.edu

I just figured out what was going on with only auto-annotating the tRNA genes.

I was working on a computer lab computer which I had set preferences on previously. But apparently DNA Master over-rode those preferences on an update at some point. Something reset the preferences to the default "Use NCBI servers".

Anyway, once I went through again and reset the server settings as listed in the page below everything worked again.

https://seaphages.org/media/docs/DNA_Master_Gene_Prediction_Settings.docx

I hope this helps save headaches for someone else. Greg

Posted in: DNA Master → Auto-annotation fix for fall 2017 and later

Link to this post \| posted 20 Feb, 2018 22:15
GregFrederick@letu.edu	RuchiraS Hey Dan, I tried this but my DNAMaster is only predicting tRNAs. This link did work before but its not working now. Thanks We are getting this same result today. It occurred both earlier this AM and again this afternoon. Are the servers down again today????

Posted in: DNA Master → Auto-annotation fix for fall 2017 and later

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