TSP Scrum

1. Targeted Sequencing Pipeline
2. Targeted Sequencing Pipeline pipeline features pipeline requirements discovery mode how does it work improvements common problems
3. sequencing Targeted Sequencing Pipeline Output structure
4. sequencing Targeted Sequencing Pipeline Output structure /share/lustre/archive/MiSeq/MiSeq_Analysis_Files/<run_id>/
5. Targeted Sequencing Pipeline Features * Alignment using bwa-mem * alignment problems * improvements * Xenograft project showed some limitations * Binomial exact test * Background size * Code working on decent coverage * No tests yet * Variant tagging needs some more work
6. Targeted Sequencing Pipeline git clone http://YOU@repo.mo.bccrc.ca/scm/pp/miseq-pipeline.git cd miseq-pipeline/pipeline/code ! python pipeline.py \ —num_cpus 10 \ —mode cluster \ —install_dir <path_to_miseq>/software \ <path_to_config_file> Limitations : ! - Manifest file - positions format - limited to point mutations ! ! If you want to run this version : ! - create_config_file_for_miseqpipeline.py One single code that generates all needed formats to run the pipeline
7. Targeted Sequencing Pipeline discovery mode
8. Input processing Alignment recalibration Variant calling and filtering Variant characterization
9. Input processing Alignment recalibration Variant calling and filtering Variant characterization * Fastq QC (not yet implemented) * Alignment (BWA-MEM, bwa) * (Bowtie2 to test ?)
10. 6% > 200 bp garbage-in garbage-out
11. Input processing Alignment recalibration Variant calling and filtering Variant characterization * Recalibrate Base Quality from sequencers * create targets for local realignments (indels) * local realignment (correction of small fraction of the alignment) * can be time consuming depending on the depth Broad best practices information : http://goo.gl/8sRWCF * Fastq QC * Alignment (BWA-MEM, bwa) * (Bowtie2 to test ?)
12. Input processing Alignment recalibration Variant calling and filtering Variant characterization * Recalibrate Base Quality from sequencers * create targets for local realignments (indels) * local realignment (correction of small fraction of the alignment) * can be time consuming depending on the depth Broad best practices information : http://goo.gl/8sRWCF * Fastq QC * Alignment (BWA-MEM, bwa) * (Bowtie2 to test ?) * call variants (any caller), I tested UnifiedGenotyper and HaplotypeCaller * Merge all vcfs for all the samples * Intersect with amplicons positions and filter out call out of the targets * Get unique calls across all samples
13. Input processing Alignment recalibration Variant calling and filtering Variant characterization * Recalibrate Base Quality from sequencers * create targets for local realignments (indels) * local realignment (correction of small fraction of the alignment) * can be time consuming depending on the depth Broad best practices information : http://goo.gl/8sRWCF * Fastq QC * Alignment (BWA-MEM, bwa) * (Bowtie2 to test ?) * call variants (any caller), I tested UnifiedGenotyper and HaplotypeCaller * Merge all vcfs for all the samples * Intersect with amplicons positions and filter out call out of the targets * Get unique calls across all samples * Build counts file per sample * Create foreground and background * binomial test and tag the variant * report generation
14. Targeted Sequencing Pipeline git clone <stash>/scm/~raniba/targeted_sequencing_pipeline.git! cd targeted_sequencing_pipeline! git checkout dev! ! python pipeline.py \! ! ! --num_cpus 40 \! ! ! --mode cluster \! ! ! --install_dir <path_to_tsp>/software/ \! ! ! <path_to_config.yaml> \! ! ! --dedup no \! ! ! --targets <path_to_amplicon_targets>! ! ! --email your@email Installation instructions on the README.md of the repo
15. Targeted Sequencing Pipeline |-- log |-- results `-- tmp |-- amplicon_positions |-- bam |-- calls |-- counts |-- intervals |-- merged_calls |-- positions |-- realigned |-- reheaded `-- variant_status results
16. Targeted Sequencing Pipeline