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- To use Freebayes, import your preprocessed alignment output data in BAM format and reference genome in fasta format.
- *Resources: https://github.com/ekg/freebayes
Test data for this app appears directly in the Discovery Environment in the Data window under Shared With Me” -> luj -> variant_calling.
- Use directory picard_preprocess_output from the directory above as input directory of preprocessed bam file.
- Use ricen_Chr12.fa as reference genome fasta file.
- Use ricen_Chr12_target.bed as bed file of targets
Parameters Used in App
When the app is run in the Discovery Environment, use the following parameters with the above input directory to get the output provided in the next section below.
- Use these parameters within the DE app interface:
- Check "Use stringent input base and mapping quality filters"
- The preprocessed bam file should be bam file that has been sorted, deduped, and read group info added.
- You could limit the analysis to target regions in a bed file
- You could provide known variants, such as dbsnp, in a vcf file
- You could set following parameters of analysis
- Using stringent filter, it is the same as -0 (standard filters: --min-mapping-quality 30 --min-base-quality 20 --min-supporting-allele-qsum 0 --genotype-varinat-threshold 0)
- The minimum number of supporting reads required (--min-coverage)
- The minimum mapping quality of read (--min-mapping-quality)
- The minimum allowed base-calling quality (--min-base-quality)
- Using Frequency-based pooled calling, it is equivalent to using FreeBayes with the following options: --haplotype-length 0 --min-alternate-count 1 --min-alternate-fraction 0 --pooled-continuous --report-monomorphic. This is the best choice for calling varinats in mixtures such as viral, bacterial, or organellar genomes.
Expect a vcf file named freebayes_output.vcf as output.