Targeted Sequencing Pipeline

Notes about the current status of the pipeline and our analyses

1. Targeted Sequencing Pipeline
2. Targeted Sequencing Pipeline pipeline features pipeline requirements discovery mode how does it work improvements common problems Results and Validation
3. Targeted Sequencing Pipeline M A N I F E S T! OR NOTHING ! WE HAVE A LOT OF DATAAAA CLASSIC VERSION DISCOVERY MODE Positions known ? Yes No TSP
4. Targeted Sequencing Pipeline M A N I F E S T! OR NOTHING ! WE HAVE A LOT OF DATAAAA CLASSIC VERSION DISCOVERY MODE Positions known ? Yes No fastq TSP
5. Targeted Sequencing Pipeline WE HAVE A LOT OF DATAAAA CLASSIC VERSION DISCOVERY MODE Positions known ? Yes No fastq TSP Job already sent … Fiction
6. Targeted Sequencing Pipeline WE HAVE A LOT OF DATAAAA CLASSIC VERSION DISCOVERY MODE Positions known ? Yes No fastq TSP I am gonna trim! these FASTQ agaiiiiin !! Reality
7. Targeted Sequencing Pipeline M A N I F E S T! OR NOTHING ! WE HAVE A LOT OF DATAAAA CLASSIC VERSION DISCOVERY MODE Positions known ? Yes No fastq TSP Bam Count Binomial Dependencies Coverage Allele frequencies log log log !! qsub drmaa rocks down rocks down
8. Targeted Sequencing Pipeline M A N I F E S T! OR NOTHING ! WE HAVE A LOT OF DATAAAA CLASSIC VERSION DISCOVERY MODE Positions known ? Yes No fastq TSP Bam Count Binomial Dependencies Coverage Allele frequencies log log log !! qsub drmaa rocks down rocks down rsync Excel Feedback quality
9. Targeted Sequencing Pipeline M A N I F E S T! OR NOTHING ! WE HAVE A LOT OF DATAAAA CLASSIC VERSION DISCOVERY MODE Positions known ? Yes No fastq TSP Bam Count Binomial Dependencies Coverage Allele frequencies log log log !! qsub drmaa rocks down rocks down rsync Excel Feedback quality
10. sequencing Targeted Sequencing Pipeline Output structure
11. sequencing Targeted Sequencing Pipeline Output structure /share/lustre/archive/MiSeq/MiSeq_Analysis_Files/<run_id>/
12. 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
13. 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
14. 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>
15. Targeted Sequencing Pipeline discovery mode
16. Input processing Alignment recalibration Variant calling and filtering Variant characterization
17. Input processing Alignment recalibration Variant calling and filtering Variant characterization * Fastq QC (not yet implemented) * Alignment (BWA-MEM, bwa) * (Bowtie2 to test ?)
18. 6% > 200 bp garbage-in garbage-out
19. 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 ?)
20. 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
21. 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
22. Targeted Sequencing Pipeline git clone <stash>! 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
23. Targeted Sequencing Pipeline git clone python pipeline.py \! ! ! ! ! ! ! --install_dir <path_to_tsp> ! ! <path_to_config.yaml> ! ! ! ! --targets ! ! -- Installation instructions on the
24. Targeted Sequencing Pipeline Validation Xenograft SA495
25. Targeted Sequencing Pipeline Validation Xenograft SA495 classic-bwa classic-bowtie2 discovery-mode
26. Targeted Sequencing Pipeline Aligners : test, compare and pick classic-bwa NSG
27. Targeted Sequencing Pipeline Aligners : test, compare and pick NSG 86 % classic-bwa
28. Targeted Sequencing Pipeline Aligners : test, compare and pick classic-bowtie2 NSG
29. Targeted Sequencing Pipeline Aligners : test, compare and pick NSG 97 % classic-bowtie2
30. Targeted Sequencing Pipeline Aligners : test, compare and pick discovery-mode NSG
31. Targeted Sequencing Pipeline Aligners : test, compare and pick NSG 97 % discovery-mode
32. Targeted Sequencing Pipeline Coverage
33. Targeted Sequencing Pipeline Coverage
34. Targeted Sequencing Pipeline Coverage
35. Targeted Sequencing Pipeline Allelic Frequencies
36. Targeted Sequencing Pipeline Allelic Frequencies
37. Targeted Sequencing Pipeline Allelic Frequencies
38. Targeted Sequencing Pipeline Allelic Frequencies
39. Targeted Sequencing Pipeline Allelic Frequencies
40. Targeted Sequencing Pipeline - Bugs found and fixed in the original codebase - Classic version on Stash (branch rad) - Bowtie over BWA (Single Cell) - Discovery mode (++features) - mutation-seq deep seq model - Discovery mode on Stash ! ! - Classic version Factory based version : Done and being tested - Add components for the discovery mode
41. Targeted Sequencing Pipeline