.. image:: ../../images/pVACseq_logo_trans-bg_sm_v4b.png :align: right :alt: pVACseq logo .. _phased_vcf: Creating a phased VCF of proximal variants ========================================== By default, pVACseq will evaluate all somatic variants in the input VCF in isolation. As a result, if a somatic variant of interest has other somatic or germline variants in proximity, the calculated wildtype and mutant protein sequences might be incorrect because the amino acid changes of those proximal variants were not taken into account. To solve this problem, we added a new option to pVACseq in the ``pvactools`` release 1.1. This option, ``--phased-proximal-variants-vcf``, can be used to provide the path to a phased VCF of proximal variants in addition to the normal input VCF. This VCF is then used to incorporate amino acid changes of nearby variants that are in-phase to a somatic variant of interest. This results in corrected mutant and wildtype protein sequences that account for proximal variants. At this time, this option only handles missense proximal variants but we are working on a more comprehensive approach to this problem. Note that if you do not perform the proximal variants step, you should manually review the sequence data for all candidates (e.g. in IGV) for proximal variants and either account for these manually, or eliminate these candidates. Failure to do so may lead to inclusion of incorrect peptide sequences. How to create the phased VCF of proximal variants ------------------------------------------------- Input files ___________ - ``tumor.bam``: A BAM file of tumor reads - ``somatic.vcf``: A VCF of somatic variants - ``germline.vcf``: A VCF of germline variants - ``reference.fa``: The reference FASTA file Required tools ______________ - `Picard `_ - `GATK `_ - `bgzip `_ - `tabix `_ Create the reference dictionary _______________________________ .. code-block:: none java -jar picard.jar CreateSequenceDictionary \ R=reference.fa \ O=reference.dict Extract a tumor-only VCF from the somatic VCF _____________________________________________ .. code-block:: none /usr/bin/java -Xmx4g -jar /opt/GenomeAnalysisTK.jar \ -T SelectVariants \ -R reference.fa --variant somatic.vcf --sample-name tumor_sample_name --output tumor_only.vcf Index the tumor-only VCF ________________________ .. code-block:: none tabix -p vcf tumor_only.vcf Update sample names ___________________ The sample names in the ``tumor.bam``, the newly-created ``tumor_only.vcf``, and the ``germline.vcf`` need to match. If they don't you need to edit the sample names in the VCF files to match the tumor BAM file. Combine somatic and germline variants using GATK’s CombineVariants __________________________________________________________________ .. code-block:: none /usr/bin/java -Xmx16g -jar /opt/GenomeAnalysisTK.jar \ -T CombineVariants \ -R reference.fa \ --variant germline.vcf \ --variant tumor_only.vcf \ -o combined_somatic_plus_germline.vcf \ --assumeIdenticalSamples Sort combined VCF using Picard ______________________________ .. code-block:: none /usr/bin/java -Xmx16g -jar /opt/picard/picard.jar SortVcf \ I=combined_somatic_plus_germline.vcf \ O=combined_somatic_plus_germline.sorted.vcf \ SEQUENCE_DICTIONARY=reference.dict Phase variants using GATK’s ReadBackedPhasing _____________________________________________ Unfortunately, the tool used for this step is no longer available in current versions of GATK. We recommend using GATK 3.6.0 to run this step. .. code-block:: none /usr/bin/java -Xmx16g -jar /opt/GenomeAnalysisTK.jar \ -T ReadBackedPhasing \ -R reference.fa \ -I tumor.bam \ --variant combined_somatic_plus_germline.sorted.vcf \ -L combined_somatic_plus_germline.sorted.vcf \ -o phased.vcf Annotate VCF with VEP _____________________ .. code-block:: none ./vep \ --input_file --output_file \ --format vcf --vcf --symbol --terms SO --tsl\ --hgvs --fasta \ --offline --cache [--dir_cache ] \ --plugin Downstream --plugin Wildtype \ [--dir_plugins ] [--pick] [--transcript_version] .. _bgzip_phased_vcf: bgzip and index the phased VCF ______________________________ .. code-block:: none bgzip -c phased.annotated.vcf > phased.annotated.vcf.gz tabix -p vcf phased.annotated.vcf.gz The resulting ``phased.vcf.gz`` file can be used as the input to the ``--phased-proximal-variants-vcf`` option. .. _bgzip_input_vcf: bgzip and index the pVACseq main input VCF __________________________________________ In order to use the ``--phased-proximal-variants-vcf`` option you will also need to bgzip and index the VCF you plan on using as the main input VCF to pVACseq. This is usually the same somatic.vcf used as input for creating the phased proximal VCF after all the required and desired optional preprocessing steps (e.g. VEP annotation, adding readcount and expression data) have been done on that VCF. Bgzipping and indexing the fully pre-processed somatic VCF is the last step. .. code-block:: none bgzip -c input.vcf > input.vcf.gz tabix -p vcf input.vcf.gz