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Developed in the Data Sciences Platform at the Broad Institute, the toolkit offers a wide variety of tools with a primary focus on variant discovery and genotyping. Its powerful processing engine and high-performance computing features make it capable of taking on projects of any size. Learn more

Data pre-processing for variant discovery - BaseRecalibrator, vcf input?

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    Genevieve Brandt

    Hi Arik, which files do you have and which are you missing? Please provide the specific command

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    Arye Harel

    Hi Genevieve, 

    Thank you for your fast response.
    I am missing a vcf file.
    These are the main stages and files I already ran:

    1. Task: QA
    Software: fastqc
    Input:   individualA_f.fq.gz , individualA_r.fq.gz derived from illumina Hiseq
    Result: 2 html files showing sequences with duplicates and adapters.


    2. Task: remove low QA sequences, and adapters.
    Software: Trimmomatic 
    input:  individualA_f.fq.gz , individualA_r.fq.gz
    output: individualA_f_paired.fq.gz , individualA_r_paried.fq.gz
                 individualA_f_unpaired.fq.gz , individualA_r_unparied.fq.gz

    ---- From here on protocol is based on GATK  ---

    3. Mapping (based on GATK)

    3A. Indexing:

    Command: bwa index -a bwtsw Genome.fasta
    input:  PlantGenome.fasta
    output: indexing files for genome 

    3B. Mapping:

    Command: bwa mem -M -t 14 PlantGenome.fasta individualA_f_paired.fq , individualA_r_paried.fq > individualA_paired.sam

    input:  see in command
    output: individualA_paired.sam

    4. Task: Mark Duplicates

    4a. sort
    Command: picard.jar SortSam I=individualA_paired.sam  O=individualA_paired_sorted.sam SORT_ORDER=coordinate

    input: 
    see in command
    output: individualA_paired_sorted.sam

    4b. markduplicates
    Command: picard.jar MarkDuplicates  I=individualA_paired_sorted.sam O=individualA_paired_markDup.sam ASSUME_SORT_ORDER=coordinate M=individualA_markDuplic_matrix

    input:  see in command
    output: individualA_paired_markDup.sam, individualA_markDuplic_matrix

    4c. sort again

    Same command as in 4a:
    input:  individualA_paired_markDup.sam
    output: individualA_paired_markdup_ReSorted.sam

     

    5. The next staage according to https://gatk.broadinstitute.org/hc/en-us/articles/360035535912-Data-pre-processing-for-variant-discovery is:

    Base (Quality Score) Recalibration done with BaseRecalibrator and Apply Recalibration.

    However BaseRecalibrator requires this argument:
    --known-sites sites_of_variation.vcf

    However, I still don't have a vcf file at this stage of the protocol.

    In other words: how do I make the  sites_of_variation.vcf required by the --known-sites argument? In which stage it should have been created?

    What am I am missing here?

     

    Thank you very much for your hep,

     

    Arik

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    Arye Harel

    One more question:

    Looking at "https://gatk.broadinstitute.org/hc/en-us/articles/360035535912-Data-pre-processing-for-variant-discovery", it reads:

    "Tools involved: BaseRecalibrator, Apply Recalibration, AnalyzeCovariates (optional)"

    Does it mean the whole "Base (Quality Score) Recalibration" stage is  an optional stage? Or  just the AnalyzeCovariates stage?

    If the whole stage is optional when does one removes duplicates? in the MarkDuplicates stage?

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    Genevieve Brandt

    Hello Arik, the Base Quality Score Recalibration (BQSR) step is recommended to improve results, but not required to run an analysis using GATK. You need at least 100M bases per read group to get high quality results. Here is an overview in our documentation: Base Quality Score Recalibration (BQSR)

    The file: --known-sites sites_of_variation.vcf is not created in your workflow, it is a VCF file containing the variants that have been confirmed for the species you are studying. Here is more information about where you can find those files: Where can I find known variants, training and truth sets, and other resource files

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    Arye Harel

    Dear Genevieve,

    Thank you very much for the helpful response.

    Arik

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    Genevieve Brandt

    Glad we were able to help!

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