This workflow carries out a Germline Exome Analysis pipeline, including the integration of variants from various callers and basic annotation.
1) RNA Alignments are then recalibrated and realigned using GATK3 (DePristo et al 2011;McKenna et al 2010)
2) To detect genome germline variants, GATK3 (DePristo et al 2011, McKenna et al 2010), Platypus (Rimmer et al 2014), Samtools version 1.3 and FreeBayes version 0.9.7 (Garrison and Marth 2012) are used.
3) Integration of predicted SNPs and INDELs from these algorithms is performed using BAYSIC (Cantarel et al 2014).
4) Effect of SNPs and INDELs on genes is predicted using snpEff (Cingolani et al 2012) using the gencode gene annotations. For GRCH38 Only: allele frequency in the general population is determined by comparison to ExAC (The ExAC Consortium 2015). Additionally for this build, discovered variants are annotated using SnpSift (Cingolani et al 2012) using the dbSNP, COSMIC (Forbes et al. 2009), CLINVAR (Landrum et al 2014), GWAS Catalog (Welter et al 2014) and dbNSFP (Liu et al 2011) databases.
5) Features (genes, transcripts and exons) are counted using featureCounts (Liao et al 2014) using the Gencode feature table(Harrow et al. 2012)
##Workflow Parameters
rnabam - Choose the alignments of your RNASeq data (generated by RNASEq Differential Expression Pipeline).
dnabam - Choose the bamfiles from genomic data that should be used for gene fusion
genome - Choose a genomic reference (genome).
pairs - Choose if pair-ended or single-end sequences
incdna - Choose whether GeneFusion analysis should include evidence from genomic data from the same sample
design - This file matches the fastq files to data about the sample
The following columns are necessary, must be named as in template and can be in any order:
SampleID
This ID should match the name in the fastq file ie S0001.R1.fastq.gz the sample ID is S0001
SampleName
This ID can be the identifier of the researcher or clinician
SubjectID
Used in order to link samples from the same patient
Phenotype
2= Case or Diseaes Phenotype, 1= Healthy Control
Gender
1=male, 2=female
FullPathToFqR1
Name of the fastq file R1
FullPathToFqR2
Name of the fastq file R2
There are some optional columns that might help with the analysis:
SequenceRun
Organism
FamilyID
CellPopulation
Treatment
GeneticFeature (WT or KO)
Race
Ethnicity
Age
### Test Data
### Credits
This example worklow is derived from original scripts kindly contributed by the Bioinformatic Core Facility (BICF), Department of Bioinformatics and Clinical Sciences.
### References
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Cantarel, B. L., Weaver, D., McNeill, N., Zhang, J., Mackey, A. J., & Reese, J. (2014). BAYSIC: a Bayesian method for combining sets of genome variants with improved specificity and sensitivity. BMC Bioinformatics, 15, 104. doi:10.1186/1471-2105-15-104
Cingolani, P., Platts, A., Wang, L. L., Coon, M., Nguyen, T., Wang, L., ? Ruden, D. M. (2012). A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly. doi:10.4161/fly.19695
Cingolani, P., Patel, V. M., Coon, M., Nguyen, T., Land, S. J., Ruden, D. M., & Lu, X. (2012). Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift. Frontiers in Genetics. doi:10.3389/fgene.2012.00035
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THIS WORKFLOW IS OBSOLETE! The Main BICF workflow includes variant analysis and differential expression analysis as one easy to use workflow.
