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
 * 3) Genomes > Create .genome
 * 4) * Menu: Genomes > Create .genome file
 * 5) * Unique Identifier: NC_006998.1
 * 6) * Descriptive Name: VACV-WR
 * 7) * FASTA File: NC_006998.fna
 * 8) * Gene File: NC_006998.gff

Ref-based Protocol

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

Workflow for this protocol: (import to use) Example History:
 * 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
 * https://galaxy.uabgrid.uab.edu/u/curtish/h/gbs722-vacv-wr-125k---sample-bwa-to-ref-in-history

Denovo Protocol (2x)

 * 1) Denovo contigs :
 * 2) NGS: Assembly > velveth (k=29, format=fastq, shortPairedRead & shortPairedRead2)
 * 3) NGS: Assembly > velvetg (MinContigLen=200, UsePairedReads=250)
 * 4) Map Contigs to Ref:
 * 5) Demo Tools > BWA SW for Contigs (ref from history)
 * 6) SAM Tools > SAM-to-BAM (ref from history)
 * 7) rename & download .bam & .bai for IGV visualization
 * 8) QC:
 * 9) SAM Tools > flagstat
 * 10) BWA_SW SAM un-mapped contigs:
 * 11) http://ncbi.nlm.nih.gov > nucleotide blast megablast

Workflow for this protocol: (import to use) Example History:
 * Shared Data > Workflows > GBS722 VACV-WR 125k - sample denovo with BWASW to ref in hist
 * https://galaxy.uabgrid.uab.edu/u/curtish/w/gbs722-vacv-wr-125k---sample-denovo-with-bwasw-to-ref-in-hist
 * https://galaxy.uabgrid.uab.edu/u/curtish/h/gbs722-vacv-wr-125k---sample-bwa-to-ref-in-history

Recommended Visualization

 * 1) IGV, load tracks
 * 2) WT BWA_BAM
 * 3) Drug BWA_BAM
 * 4) WT Velvet_BWASW_BAM
 * 5) Drug Velvet_BWASW_BAM