Analysis:Radish dCAPS

From RadishDB

Contents 1 Planning 2 SNP alignment file processing 3 dCAPS finding tools 3.1 Potential Tools 3.2 Testing 3.2.1 SNP2CAPS 3.2.2 dCAPS Finder 4 Continue on SNP2CAPS 4.1 SNP2CAPS script dev 4.2 EMBOSS restrict 5 Enzyme set II

Planning

• Dworkin and Shim have worked out a pipeline.

SNP alignment file processing

• Get the SNP position with the frequency of 2nd most abundant nucleotide >= 3 and get flanking 10bp

python ~/project/radish/_script/script_6.1_parse_align.py align_snp.all 3 10

dCAPS finding tools

Potential Tools

• dCAPS Finder
• SNP2CAPS

Testing

SNP2CAPS

• Get relevant files: code, example, and rebase.

wget http://pgrc.ipk-gatersleben.de/snp2caps/download/SNP2CAPS.pl
wget http://pgrc.ipk-gatersleben.de/snp2caps/download/example
wget http://rebase.neb.com/rebase/link_gcg (doesn't work, need to download directly)

• This tool needs enzyme specified, not optimal.
• The downloaded perl script has some problem. It is modified and worked now.

dCAPS Finder

• Web-based, considering writing a robot.
  • Find out how to get the cgi form fields: Html form to Email
    • Original
    • File modified: change the action field to 'mailto:you@yourdomain.com'
    • Form result

seq1=ATGATAAGAGGCGGA&mutseq=ATGATAAGAGGCGGA&nmismatch=0

• Too slow.

Continue on SNP2CAPS

• The perl script does not work. Some minor modification.
• Work on:
  1. A batch SNP2CAPS run script and a parser for the output
  2. A batch EMBOSS:restrict run script and an output parser

SNP2CAPS script dev

• WD: ~/project/radish/6_snp_primer/2_batch_snp2caps
• Run example

./SNP2CAPS_mod.pl example gcg.805 BfaI,ClaI,BtgI,EarI,HaeII,NsiI,Cac8I,BsiEI,DdeI,AluI,NlaIII,Tsp45I > example.out
./SNP2CAPS_mod.pl test.fa gcg.805 Cac8I > test.out

• Split the align file

python ~/project/radish/_script/script_6.2_split_align.py

• Run batch SNP2CAPS script

python ~/project/radish/_script/script_6.2_snp2caps_run.py align_snp_all.flank.1
...

EMBOSS restrict

• WD: ~/project/radish/6_snp_primer/3_restrict
• Test run restrict

~/bin/EMBOSS-4.1.0/emboss/restrict -solofragment -sequence test.fa -sitelen 4 -outfile test.restrict -rformat2 gff -enzymes BfaI,ClaI,BtgI,EarI,HaeII,NsiI,Cac8I,BsiEI,DdeI,AluI,NlaIII,Tsp45I

• Make sure no N and X are in the sequence file

python ~/project/radish/_script/script_6.3_check_N_X.py align_snp_all.seqs
Seq with N or X: 0

• Test run batch restrict script

python ../../_script/script_6.3_call_restrict.py test.flank2.caps align_snp_all.seqs

• Note that the script:
  1. Use the coordinate based on the alignment, not individual sequences
  2. Output the 5' splice junction coordinate only
  3. Do not take into account gap positions in the coordinates. So gaps are filled with X

Enzyme set II

• Provided by David Tack.
• Target enzyme: BclI,EcoRI,EcoRV,HindIII,KpnI,NdeI,PstI
• Run SNP2CAPS

pwd = /home/shiu/project/radish/6_snp_primer/2_batch_snp2caps
# Split alignment file into 8
python ~/project/radish/_script/script_6.2_split_align.py
# Convert alignment to fasta and batch run SNP2CAPS
python ~/project/radish/_script/script_6.2_snp2caps_run.py align_snp_all.flank.1 BclI,EcoRI,EcoRV,HindIII,KpnI,NdeI,PstI
...

• Run Emboss:restrict

pwd = ~/project/radish/6_snp_primer/3_restrict
python ../../_script/script_6.3_call_restrict.py align_snp_all.flank.4.caps align_snp_all.seqs BclI,EcoRI,EcoRV,HindIII,KpnI,NdeI,PstI
...

• Get sequences

pwd = ~/project/radish/6_snp_primer/3_restrict/2_enz_set2
python ~/project/radish/_script/script_6.3_get_contig_names.py all.flank_rsites
python ~/project/radish/_script/script_6.3_select_contig_seq.py all.flank_rsites.qualified ../align_snp_all.seqs