Difference between revisions of "BZIP study"

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(Confirming domain presence)
 
Line 99: Line 99:
 
hitdata.tsv contains ''domains'', whereas featdata.tsv contains ''types of regions'' (e.g. DNA-binding).
 
hitdata.tsv contains ''domains'', whereas featdata.tsv contains ''types of regions'' (e.g. DNA-binding).
  
1. From hitdata.tsv, exclude any genes that does not have 'bZIP' in the domain description
+
1. From hitdata.tsv, exclude any genes that does not have 'bZIP' or 'BRLZ (SMART accession)' in the domain description
  
 
  python getbzip.py hitdata.tsv hitdata.bzip.txt
 
  python getbzip.py hitdata.tsv hitdata.bzip.txt
  
'''73 queries''' detected in hitdata.bzip.txt
+
'''74 queries''' detected in hitdata.bzip.txt
  
2.
+
2. Extract description, accession information from hitdata.tsv
 +
 
 +
python extractbzip.py hitdata.tsv hitdata.bzip.txt hitdata.bzip.tsv
 +
python getaccession.py hitdata.bzip.tsv hitdata.bzip.accession
 +
 
 +
* hitdata.bzip.accession
 +
 
 +
Gene_ID    Domain_start    Domain_end Domain_length    Accession    Description
 +
Vradi01g12230.1 63      108    46      cd14703 bZIP_plant_RF2
 +
-      63      108    46      cl21462 bZIP
 +
-      66      108    43      cd14704 bZIP_HY5-like
 +
-      66      107    42      cd14707 bZIP_plant_BZIP46
 +
-      63      107    45      cd14702 bZIP_plant_GBF1

Latest revision as of 07:19, 22 April 2019

Contents

Identification

Accession IDs for bZIP transcription factor domain:

  1. Pfam: PF00170
  2. SMART: SM00338
  3. Interpro: IPR004827
  4. PubMed (NCBI): PUBMED:7780801


Pfam-based approach

HMM domain clans

Protein domain information obtained from Pfam database.

Regarded as the main domain for bZIP transcription factor. Clearly distinguishes basic region and leucine zipper.

Used for further analysis.

Conserved basic region, weaker leucine zipper.

  • bZIP_Maf (PF03131)

No distinct basic region. No clear leucine zipper interface.

Downloading and generating HMM consensus sequence file

WD: 63:/data6/chojam96/bZIP/identification
  • Download Stockholm alignment file from Pfam bZIP_1 > Alignment > Download options > Seed

Seed: the curated alignment from which the HMM for the family is built.

  • Use 'hmmbuild' command from HMMER software to convert Stockholm alignment into a profile HMM

After gunzipping, use following command.

hmmbuild [options] hmmfile alignfile

hmmbuild reads a multiple sequence alignment file alignfile , builds a new profile HMM, and saves the HMM in hmmfile.

For following commands for HMMER, refer to this page.

hmmbuild PF00170.hmm PF00170.seed

hmmscanning the protein sequence

  • Use 'hmmpress' command for preparing HMM database
hmmpress [options] hmmfile

This produces four preparation files.

  • Run 'hmmscan' command against Vigna radiata pepetide sequence fasta file
hmmscan [options] hmmdb seqfile
hmmscan --tblout PF00170.out PF00170.hmm Vradi_ver6.fa.cds.primary.fasta.pepshorten.fa

No primary options used. Peptide file used for hmmscanning had only main transcripts(*.1, n=22427).

PF00170.out (result file of hmmscan) contained 147 nonredundant genes.

Out of 147 genes, 144 genes had complete peptide sequence, marked by termination codon sign (*).

  • Get corresponding protein sequences
python getgenes.py PF00170.out Vradi_ver6.fa.cds.primary.fasta.pepshorten.fa bZIP.primary.fa

Confirming domain presence

Trickest part in identification of bZIP genes - the best way is to pick and choose gene by gene, with measures.


Align peptide sequence file (bZIP.primary.fa, n=147) by 'MUSCLE' to manually see the alignment

muscle -in bZIP.primary.fa -out bZIP.primary.aligned.fa

Toggled sequence at 50% level to check conserved domains, but no consensus found.


Use 'interproscan' and 'NCBI CD search' for domain detection

  • Interproscan

19.04.21 → search ID: QM3-qcdsearch-1F885D58AB029684

Two types of results: hitdata.tsv, featdata.tsv

hitdata.tsv contains 140 genes, and 7 remaining genes confirmed as queries 'with no domain hits'

hitdata.tsv contains domains, whereas featdata.tsv contains types of regions (e.g. DNA-binding).

1. From hitdata.tsv, exclude any genes that does not have 'bZIP' or 'BRLZ (SMART accession)' in the domain description

python getbzip.py hitdata.tsv hitdata.bzip.txt

74 queries detected in hitdata.bzip.txt

2. Extract description, accession information from hitdata.tsv

python extractbzip.py hitdata.tsv hitdata.bzip.txt hitdata.bzip.tsv
python getaccession.py hitdata.bzip.tsv hitdata.bzip.accession
  • hitdata.bzip.accession
Gene_ID    Domain_start    Domain_end Domain_length    Accession    Description
Vradi01g12230.1 63      108     46      cd14703 bZIP_plant_RF2
-       63      108     46      cl21462 bZIP
-       66      108     43      cd14704 bZIP_HY5-like
-       66      107     42      cd14707 bZIP_plant_BZIP46
-       63      107     45      cd14702 bZIP_plant_GBF1