GBS722 2013: Difference between revisions
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== Ref-based Protocol == | == Ref-based Protocol == | ||
# '''QC''': Demo Tools > FastQC ( | # '''For each sample (2x)''' | ||
# '''Mapping | ## '''QC''': Demo Tools > FastQC (2x: forward & reverse reads) | ||
## Demo Tools > BWA for Illumina (ref from history, paired end) | ## '''Mapping''': | ||
## SAM Tools > SAM-to-BAM (ref from history) | ### Demo Tools > BWA for Illumina (ref from history, paired end) | ||
## rename & download .bam & .bai for IGV visualization | ### SAM Tools > SAM-to-BAM (ref from history) | ||
# '''QC''': SAM Tools > Flag Stat | ### rename & download .bam & .bai for IGV visualization | ||
# '''SNP calling''': NGS: SAM Tools > Generate VCF with mpileup piped through bcftools view | ### '''QC''': SAM Tools > Flag Stat | ||
# '''SNP assessment''': Demo Tools > SnpEff (Vaccinia_Western_Reserve (vacwr.NC_006998.1)) | ## '''SNP calling''': NGS: SAM Tools > Generate VCF with mpileup piped through bcftools view (ref from history) | ||
## '''SNP assessment''': Demo Tools > SnpEff (output=VCF; ref=Vaccinia_Western_Reserve (vacwr.NC_006998.1), up/down=200 bases, filter=select_all) | |||
# '''Compare Genomes''': Join, Subtract and Group > Compare two Datasets (columns 1-5) | # '''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) == | == Denovo Protocol (2x) == | ||
# '''Denovo contigs ''': | # '''Denovo contigs ''': | ||
## NGS: Assembly > velveth (shortPairedRead & shortPairedRead2) | ## NGS: Assembly > velveth (k=29, format=fast'''q''', shortPairedRead & shortPairedRead2) | ||
## NGS: Assembly > velvetg (MinContigLen=200, UsePairedReads=250) | ## NGS: Assembly > velvetg (MinContigLen=200, UsePairedReads=250) | ||
# '''Map Contigs to Ref''': | # '''Map Contigs to Ref''': | ||
## Demo Tools > BWA SW for Contigs | ## Demo Tools > BWA SW for Contigs (ref from history) | ||
## SAM Tools > SAM-to-BAM | ## SAM Tools > SAM-to-BAM (ref from history) | ||
## rename & download .bam & .bai for IGV visualization | ## rename & download .bam & .bai for IGV visualization | ||
# ''' | ## '''QC''': | ||
### SAM Tools > flagstat | |||
### BWA_SW SAM un-mapped contigs: | |||
#### http://ncbi.nlm.nih.gov > [http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome nucleotide blast megablast] | |||
Workflow for this protocol: (import to use) | |||
* 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 | |||
Example History: | |||
* https://galaxy.uabgrid.uab.edu/u/curtish/h/gbs722-vacv-wr-125k---sample-bwa-to-ref-in-history | |||
== Recommended Visualization == | == Recommended Visualization == | ||
Latest revision as of 18:43, 14 March 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
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
- For each sample (2x)
- QC: Demo Tools > FastQC (2x: forward & reverse reads)
- Mapping:
- 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: NGS: SAM Tools > Generate VCF with mpileup piped through bcftools view (ref from history)
- SNP assessment: Demo Tools > SnpEff (output=VCF; ref=Vaccinia_Western_Reserve (vacwr.NC_006998.1), up/down=200 bases, filter=select_all)
- 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:
Denovo Protocol (2x)
- Denovo contigs :
- NGS: Assembly > velveth (k=29, format=fastq, shortPairedRead & shortPairedRead2)
- NGS: Assembly > velvetg (MinContigLen=200, UsePairedReads=250)
- Map Contigs to Ref:
- Demo Tools > BWA SW for Contigs (ref from history)
- SAM Tools > SAM-to-BAM (ref from history)
- rename & download .bam & .bai for IGV visualization
- QC:
- SAM Tools > flagstat
- BWA_SW SAM un-mapped contigs:
Workflow for this protocol: (import to use)
- 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
Example History:
Recommended Visualization
- IGV, load tracks
- WT BWA_BAM
- Drug BWA_BAM
- WT Velvet_BWASW_BAM
- Drug Velvet_BWASW_BAM