Exercise 4

CBS >> CBS Courses >> Comparative Microbial Genomics >> Course Programme >> Exercise 4

OBJECTIVES

To get familiar with methods for assigning protein function by using Hidden Markov Models.

LOG IN
Open a session on 'organism.cbs.dtu.dk', and from this login to ibiology and create directories for you to work in:
```
ssh -X ibiology
mkdir Ex4
cd Ex4
mkdir source
```

PREDICTING SIGMA FACTORS

We will start out by using an HMM to query the proteins that you have predicted in Exercise 1. We have prepared three HMMs from alignments of three types of Sigma factors, σ 54, σ 70, and ECF. The HMMs you are going to use have been calibrated in such a way, that alignment scores greater than zero are considered significant (or at least better than the NULL model)

You will be using the database table oslo_genomics.features to store your category asignments (by using the 'note' field.

But we will start out by generating hmmsearch reports of the protein files you generated in Exercise 3

foreach accession (AE017042 AE016879 AL111168 AL645882 AP008232 AP009048 BA000021 CP000034)
   foreach type (s54 s70 ecf)
     /usr/cbs/tools/lib/make/bin/hmmsearch.Linux /usr/cbs/tools/lib/make/kiil/HMMsigma/$type.hmm \
         ../Ex3/source/$accession.proteins.fsa > source/$accession.proteins.$type.sigmas.hmmsearch &
   end
end
wait

How many HMM searches have you initiated? Try first and view the content of a hmmsearch report ( use spacebar to scroll and 'q' to quit ). In this report, you should find one protein with a score better than zero:

less source/AE017042.proteins.ecf.sigmas.hmmsearch

Ok. Now you have the hmmsearch reports, but you need to extract the feature names. The following command will filter the report and extract the gene names of the significant results and place a comment in the database table.

mysql -B -e "update oslo_genomics.features set note = '' where user = USER() and note not like 'tcs%' or note is null"
foreach accession (AE017042 AE016879 AL111168 AL645882 AP008232 AP009048 BA000021 CP000034)
   foreach type (ecf s54 s70)
     cat source/$accession.proteins.$type.sigmas.hmmsearch | \
     perl -ne 'next unless /^CDS_(\d+)\-(\d+)_DIR([\-\+]+)\s+([0-9\-\.e]+)\s+([0-9\-\.e]+)\s+/;\
               my ($start,$stop,$dir,$score,$evalue) = ($1,$2,$3,$4,$5);\
               next unless $score > 0;\
               print "update oslo_genomics.features set note = \"Sigma Factor '$type'\" \
                      where start=$start and stop = $stop and user = user() and accession = \"'$accession'\";\n";'\
     | mysql
   end
end

mysql
mysql>select start,stop,dir,note from oslo_genomics.features \
      where user=USER() and note like 'Sigma Factor%' and accession = 'CP000034' order by start;

mysql>select features.note,genomes.accession,genomes.organism,count(*) \
      from oslo_genomics.features,oslo_genomics.genomes \
      where genomes.accession=features.accession and genomes.user = features.user \
            and  features.user=USER() and note like 'Sigma Factor%' \
      group by note,genomes.accession order by note,genomes.accession;

Genome Atlas Database

Grouped by Species

mysql>select concat(genus,' ',species),count(*) as Count,avg(SIGMA54_PROMOTERS_COUNT) as AvgS54Count, \
             avg(SIGMA70_PROMOTERS_COUNT) as AvgS70Count,avg(SIGMAECF_PROMOTERS_COUNT) as AvgECFCount \
      from genomeatlas.atlasdb \
      where kingdom='bacteria' and ( segment = 'main' or segment >= 1) \
      group by genus,species;

Grouped by Phyla

mysql>select TAXLONG_PHYLA,count(*) as Count,avg(SIGMA54_PROMOTERS_COUNT) as AvgS54Count, \
             avg(SIGMA70_PROMOTERS_COUNT) as AvgS70Count,avg(SIGMAECF_PROMOTERS_COUNT) as AvgECFCount \
      from genomeatlas.atlasdb \
      where kingdom='bacteria' and ( segment = 'main' or segment >= 1) \
      group by TAXLONG_PHYLA;
mysql>exit

How does this compare with your results?

PREDICTING TWO COMPONENT SYSTEMS

The second part of todays exercise is about predicting two-component systems.

We will use the same procedure like with the sigma factors.

foreach accession (AE017042 AE016879 AL111168 AL645882 AP008232 AP009048 BA000021 CP000034)
   foreach type (RRreciever HisKA_1 HisKA_2 HisKA_3 HWE_HK)
     /usr/cbs/tools/lib/make/bin/hmmsearch.Linux --cut_tc /usr/cbs/tools/lib/make/pfh/TCS/$type.hmm \
       ../Ex3/source/$accession.proteins.fsa > source/$accession.$type.TCS.hmmsearch &
   end
end
wait

less source/AE017042.RRreciever.TCS.hmmsearch

mysql -B -e "update oslo_genomics.features set note = '' where user = USER() and note not like 'sigma%' or note is null"
foreach accession (AE017042 AE016879 AL111168 AL645882 AP008232 AP009048 BA000021 CP000034)
   foreach type (RRreciever HisKA_1 HisKA_2 HisKA_3 HWE_HK)
     cat source/$accession.$type.TCS.hmmsearch | \
     perl -ne 'next unless /^CDS_(\d+)\-(\d+)_DIR([\-\+]+)\s+([0-9\-\.e]+)\s+([0-9\-\.e]+)\s+/;\
               my ($start,$stop,$dir,$score,$evalue) = ($1,$2,$3,$4,$5);\
               next unless $score > 0; \
               print "update oslo_genomics.features set note = \"TCS '$type'\" \
                      where start=$start and stop = $stop and user = user() and accession = \"'$accession'\";\n";'\
     | mysql -B
   end
end

mysql
mysql>select start,stop,dir,note from oslo_genomics.features where user=USER() and note like 'TCS%' and accession = 'CP000034' order by start;

mysql>select features.note,genomes.accession,organism,count(*) \
      from oslo_genomics.features,oslo_genomics.genomes \
      where  genomes.accession=features.accession and genomes.user = features.user \
             and  features.user=USER() and note like 'TCS%'  \
      group by note,genomes.accession order by genomes.accession,note;