GBS722 2013

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

• genome fasta ftp://ftp.ncbi.nih.gov/genomes/Viruses/Vaccinia_virus_uid15241/NC_006998.fna
• gene annotation ftp://ftp.ncbi.nih.gov/genomes/Viruses/Vaccinia_virus_uid15241/NC_006998.gff

Reads in UAB Galaxy

• Shared Data > Data Librarires > Tutorial Data Sets > GBS722 2013 > VACV-WR drug-control
  • VACV-WR-DRUG_1.125k.fastqsanger
  • VACV-WR-DRUG_2.125k.fastqsanger
  • VACV-WR-WT_1.125k.fastqsanger
  • VACV-WR-WT_2.125k.fastqsanger
• Or import published history
  • https://galaxy.uabgrid.uab.edu/u/curtish/h/gbs722-vacv-wr125k-data

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: (import to use)

• Shared Data > Workflows > GBS722 VACV-WR 125k - sample BWA to ref in hist
• https://galaxy.uabgrid.uab.edu/u/curtish/w/gbs722-vacv-wr-125k---sample-sample-to-ref

Example History:

• https://galaxy.uabgrid.uab.edu/u/curtish/h/gbs722-vacv-wr-125k---sample-bwa-to-ref-in-history

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 (ref from history)
   2. SAM Tools > SAM-to-BAM (ref from history)
   3. rename & download .bam & .bai for IGV visualization
   4. QC:
      1. SAM Tools > flagstat
      2. BWA_SW SAM un-mapped contigs:
         1. 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