GBS722 2013

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VACV-WR WT vs DRUG protocol in Galaxy

This is an abbreviated protocol cheat-sheet to help students through the class exercises.

Links

UAB Galaxy - https://galaxy.uabgrid.uab.edu

Ref Genome @ NCBI

Reads in UAB Galaxy

IGV Setup

  1. JNLP launch: http://www.broadinstitute.org/software/igv/download
  2. Create VACV-WR genome
    1. Genomes > Create .genome
      • Menu: Genomes > Create .genome file
      • Unique Identifier: NC_006998.1
      • Descriptive Name: VACV-WR
      • FASTA File: NC_006998.fna
      • Gene File: NC_006998.gff

Ref-based Protocol

  1. For each sample (2x)
    1. QC: Demo Tools > FastQC (2x: forward & reverse reads)
    2. Mapping:
      1. Demo Tools > BWA for Illumina (ref from history, paired end)
      2. SAM Tools > SAM-to-BAM (ref from history)
      3. rename & download .bam & .bai for IGV visualization
      4. QC: SAM Tools > Flag Stat
    3. SNP calling: NGS: SAM Tools > Generate VCF with mpileup piped through bcftools view (ref from history)
    4. SNP assessment: Demo Tools > SnpEff (output=VCF; ref=Vaccinia_Western_Reserve (vacwr.NC_006998.1), up/down=200 bases, filter=select_all)
  2. Compare Genomes: Join, Subtract and Group > Compare two Datasets (columns 1-5)

Workflow for this protocol: https://galaxy.uabgrid.uab.edu/u/curtish/w/gbs722-vacv-wr-125k---sample-sample-to-ref (import to use)

Denovo Protocol (2x)

  1. Denovo contigs :
    1. NGS: Assembly > velveth (k=29, format=fastq, shortPairedRead & shortPairedRead2)
    2. NGS: Assembly > velvetg (MinContigLen=200, UsePairedReads=250)
  2. Map Contigs to Ref:
    1. Demo Tools > BWA SW for Contigs
    2. SAM Tools > SAM-to-BAM
    3. rename & download .bam & .bai for IGV visualization
  3. NCBI Blast un-mapped contigs: http://ncbi.nlm.nih.gov > nucleotide blast megablast

Recommended Visualization

  1. IGV, load tracks
    1. WT BWA_BAM
    2. Drug BWA_BAM
    3. WT Velvet_BWASW_BAM
    4. Drug Velvet_BWASW_BAM
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