Prepare sequence for PCR verification

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Get Consensus Sequence for Primer Design

Our task is to get the consensus sequence for primer design. The steps in this task are: Note: all the input and output files should be in the fold "output" and all the perl scripts are in the fold "perl scripts".

Contents

Get the information about exons (outlier & detected) to form the Input File.

The information includes:

  1. the refseq ID of the human transcript in which the exons are located;
  2. the name of the chromosome in which the exons are located;
  3. the chain of chromosome (+/-)
  4. the ID of the outlier exon;
  5. the ID and the start/stop position in the chromosome of the exons(including one outlier and other detected exons). 
  Sample:
  REF_ID NM_000350
  CHR	chr1
  CHAIN	-
  OUTLIER	66741
  EXON_CLUSTER_ID START STOP
  66732 94230989 94231380
  66735 94234253 94234339
  66737 94236005 94236083   
  66738 94236107 94236254
  ...

You can get the positions of the exons from files named <chromosome name>_exon_location.txt. These files are produced using command: 

 perl get_exon_id_from_affymatrix.pl <chromosome name>

Extract the human transcript sequence from Human Refseq Database and find the corresponding positions of the exons in the transcript.

run perl command: 

 perl get_human_refseq_exon_position.pl <Input File>

Two files will be produced: <Refseq ID>_seq.fa and <Refseq ID>_location.txt. You should put the two files in the fold output.

Search the chimp and rhesus Refseq Database with human transcript using BLAST.

Commands: 

export PATH=$PATH:/usr/local/blastall
blastall -p blastn -d ../rna_database/rna_chimp.fa -i  <Refseq ID>_seq.fa -o <Refseq ID>_chimp.txt -m8 -b1
blastall -p blastn -d ../rna_database/rna_rhesus -i <Refseq ID>_seq.fa -o <Refseq ID>_rhesus.txt -m8 -b1

Two files will be produced: <Refseq ID>_chimp.txt and <Refseq ID>_rhesus.txt.

ps: create blast database before lauching blastall 

formatdb -p F -o F -a F -i ../rna_database/rna_human.fa
formatdb -p F -o F -a F -i ../rna_database/rna_chimp.fa
formatdb -p F -o F -a F -i ../rna_database/rna_rhesus.fa

Extract the chimp and rhesus transcript sequences from Chimp Refseq Database and Rhesus Refseq Database respectively.

Commands: If you want to align human and chimp transcrips, run this: 

perl get_seqs_for_2alignment.pl <Refseq ID>

If you wan to align three species (human, chimp and rhesus), run this: 

perl get_seqs_for_3alignment.pl <Refseq ID>

A file will be produced: <Refseq ID>_for_2alignment.fa or <Refseq ID>_for_3alignment.fa.

Align the transcripts of the species using Soft clustalw.

The Soft clustalw is in the folder clustalw1.83. The input file is <Refseq ID>_for_2alignment.fa or <Refseq ID>_for_3alignment.fa. Two file will be produced: <Refseq ID>_for_2alignment.aln or <Refseq ID>_for_3alignment.aln and <Refseq ID>_for_2alignment.dnd or <Refseq ID>_for_2alignment.dnd. You should put the first file in fold output. 

sh clustalw_for_2seq.sh <Refseq ID>

or 

sh clustalw_for_3seq.sh <Refseq ID>

Get the consensus sequence.

Command: 

 perl get_consensus_seq.pl <Refseq ID> <2/3>

A file will be produced: cons_2_<Refseq ID>.txt or cons_3_<Refseq ID>.txt. Note: the sequence of the outlier exon will be in capital characters and the start/end of the detected exon sequences will also be in capital characters.

If you want to allow gaps in the consensus sequence, you should recalculate the positions of the exons.

Command: 

perl get_gapped_position.pl <Refseq ID> <2/3>

A file will be produced: gapped_<Refseq ID>_location.txt.

Files

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