Workshop:Conner

From Shiu Lab

Contents 1 Environment 1.1 Cygwin 1.2 SSH access 2 SSR in UTRs, CDS 3 SSR mapping script 4 SSR in three pairs of closely related plant species 4.1 Species selection 4.2 Major questions 4.3 What the students did 5 SNP, CAP, dCAP 6 Intron spanning marker 7 References

Environment

Cygwin

• Cygwin is a Linux-like environment for Windows.
• Useful packages in addition to the base:
  1. openssh
  2. vim
  3. wget
  4. perl and python

SSH access

• WinSCP is a free SFTP, FTP and SCP client for Windows.
• Putty: ssh client for PC. For Mac, just use the terminal.
• Sign up for HPC

SSR in UTRs, CDS

• MISA: SSR identification script.
  • publication
  • download page
  • Test dataset: Brassica ESTs
    • NCBI dbEST
      • Search for "Brassica oleracea"
      • Search for "Raphanus"
      • Save sequences as FASTA files.
  • Run misa

perl misa.pl fasta_file_name

• Determine whether the repeats are in CDS or UTR
  • Similarity search software: BLAST
    • Build a database of protein sequences from 5 plants
    • Run ESTs against the protein database

• Compare the MISA output against the output of BLAST

SSR mapping script

• Emma is responsible for this part.
• Example files:
  • Raphanus EST sequences
  • MISA run output
  • BLAST output

SSR in three pairs of closely related plant species

Species selection

• Look into the species relationships in phytome and select species pairs based on the following criteria:
  1. Two species in the same genus or the same family.
  2. More than 10,000 EST contigs (see PlantGDB)
     • You will need the following scripts from /home/shiu/codes:
       1. BlastUtility.py
       2. ParseBlast.py
       3. FastaManager.py
       4. FileUtility.py
       5. SingleLinkage.py
       6. Translation.py
     • Download all scripts in a single zip file
     • Determine the number of EST contig by running:
       • python FastaManager.py -f count -fasta fasta_file_name
  3. Species that somehow interest you.
     • Check out what they are in NCBI Taxonomy Database

• Species we will study:

Major questions

1. What are the SSR type, freuqency, and distribution?
2. What is the level of SSR conservation cross species?
3. Are SSRs in coding sequences tend to locate in regions with high or low conservation?

What the students did

• Shiu 16:52, 18 July 2007 (EDT)

1. Identify SSRs in the EST contigs of 8 plant species.
2. Run BLAST on a two-species sequence file for identifying reciprocal best hits.
3. Run BLAST using the two-species EST sequences as queries and all plant protein sequences as subjects for identifying UTR and CDS.

SNP, CAP, dCAP

• SNP call will be done in JCVI.
• CAP finding
  • SNP to CAP: SNP2CAPS
    • publication
    • download page
    • Also need rebase
    • Also need Bioperl.
  • Test dataset: NCBI UNIGENE for Brassica napus.
    • unigene depository: download the sequence file
  • ClustalX: for sequence alignment.
  • Genedoc: Multiple sequence alignment viewer and editor.
  • New England Biolab cutter interface

• dCAP finding
  • dCAP-finder

Intron spanning marker

• Use couple examples, Arabidopsis full length cDNA.

References

• Identification of genic moss SSR markers and a comparative analysis of twenty-four algal and plant gene indices reveal species-specific rather than group-specific characteristics of microsatellites
• Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes
• Nonrandom distribution and frequencies of genomic and EST-derived microsatellite markers in rice, wheat, and barley
• Genic microsatellite markers in plants: features and applications