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
- 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
IGV Setup
- JNLP launch: http://www.broadinstitute.org/software/igv/download
- Create VACV-WR genome
- 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
- Genomes > Create .genome
Ref-based Protocol
- QC: Demo Tools > FastQC (4x)
- Mapping (2x):
- Demo Tools > BWA for Illumina (ref from history, paired end)
- SAM Tools > SAM-to-BAM (ref from history)
- rename & download .bam & .bai for IGV visualization
- QC: SAM Tools > Flag Stat
- SNP calling (2x): NGS: SAM Tools > Generate VCF with mpileup piped through bcftools view
- SNP assessment (2x): Demo Tools > SnpEff (Vaccinia_Western_Reserve (vacwr.NC_006998.1))
- Compare Genomes: Join, Subtract and Group > Compare two Datasets (columns 1-5)
Denovo Protocol (2x)
- Denovo contigs :
- NGS: Assembly > velveth (shortPairedRead & shortPairedRead2)
- NGS: Assembly > velvetg (MinContigLen=200, UsePairedReads=250)
- Map Contigs to Ref:
- Demo Tools > BWA SW for Contigs
- SAM Tools > SAM-to-BAM
- rename & download .bam & .bai for IGV visualization
- NCBI Blast un-mapped contigs: http://ncbi.nlm.nih.gov > nucleotide blast megablast
Recommended Visualization
- IGV, load tracks
- WT BWA_BAM
- Drug BWA_BAM
- WT Velvet_BWASW_BAM
- Drug Velvet_BWASW_BAM