Alternative Splicing Analysis Using PACBIO Targeted Long-Reads Sequencing Data
Description
This snakemake pipeline is for alternative splicing analysis using PACBIO targed long-reads sequencing data. The pipeline may be run on an HPC or in a local environment.
Major steps in the workflow include:
- General data processing using ISOSEQ3
- Alignment and splicing analysis of non-collaplsed reads using GMAP and SQANTI3
- Further QC filtering and splicing analysis of collapsed reads using cDNA_cupcake and SQANTI3
- Generateing customozied tappAS annotation file with sample merged data using SQANTI3
Expected results include:
- Detection and quantification of transcripts in targed regions with non-collapsed reads
- The concise version of detection of transcripts in targed regions with collapsed reads
- Customized tappAS annotation file
Software Requirements
User's guide
I. Input requirements
- Edited config/config.yaml
- Indexed bam files of demultiplexed subreads data
- Reference genome file and annotation files
II. Editing the config.yaml
Basic parameters:
- subread: Path to subread data stored directory
- targeted: Genomic coordinates of the targed region; chromosome:start_position-end_position
- ref: hg38, hg19, mm10, etc
- genome: Path to referece genome fasta file
- gtf: Path to reference annotation gtf file
- gff3: Path to reference annotation gff3 file
- tappas_gff3: Path to standard tappAS reference annotation gff3 file
- tofu: Path to the directory where the tool tofu in cDNA_cupcake package is located
- sqanti3: Path to the directory where the SQANTI3 package is located
Optional parameters:
- out: Path to desired directory to store output files, defalut: working directory
- gmap_ref: Path to GMAP index directory if available
III. To run
- Clone the repository to your working directory
git clone https://github.com/NCI-CGR/Pacbio-Alternative-Splicing.git- Install required software
- Create conda environment using the provided yaml file and activate it after installation:
conda env create -f pacbioAS_env.yaml
conda activate pacbioAS- Edit and save config/config.yaml
- To run on an HPC using slurm job scheduler: Edit config/cluster_config.yaml according to your HPC information Run run.sh to initiate running of the pipeline
- To run in a local environment:
export PYTHONPATH=$PYTHONPATH:{PATH}/cDNA_Cupcake/sequence/:{PATH}/conda/envs/sqanti3/lib/python3.7/site-packages/ snakemake -p --cores 16 --keep-going --rerun-incomplete --jobs 300 --latency-wait 120 all
- Look in log directory for logs for each rule
- To view the snakemkae rule graph:
snakemake --rulegraph | dot -T png > pacbioAS.png
IV. Example output
. user/defined/output_dir
├── ccs_bam # ccs reads
│ ├── {sample_A}_ccs.bam
│ ├── {sample_A}_ccs.bam.pbi
│ ├── {sample_A}_ccs.other_files
│ ├── {sample_B}_ccs.bam
│ ├── {sample_B}_ccs.bam.pbi
│ ├── {sample_B}_ccs.other_files
│ └── merged_ccs.bam
├── cluster # clusted unpolished reads
│ ├── {sample_A}.unpolished.bam
│ ├── {sample_A}.unpolished.bam.pbi
│ ├── {sample_A}.unpolished.cluster_report.csv
│ ├── {sample_A}.unpolished.fasta.gz
│ ├── {sample_A}.unpolished.other_files
│ ├── {sample_B}.unpolished.bam
│ ├── {sample_B}.unpolished.bam.pbi
│ ├── {sample_B}.unpolished.cluster_report.csv
│ ├── {sample_B}.unpolished.fasta.gz
│ ├── {sample_B}.unpolished.other_files
│ ├── merged.unpolished.bam
│ ├── merged.unpolished.bam.pbi
│ ├── merged.unpolished.cluster_report.csv
│ ├── merged.unpolished.fasta.gz
│ └── merged.unpolished.other_files
├── collapsed # collapsed reads
│ ├── {sample_A}.collapsed.abundance.txt
│ ├── {sample_A}.collapsed.fasta
│ ├── {sample_A}.collapsed.filtered.abundance.txt
│ ├── {sample_A}.collapsed.filtered.rep.fasta
│ ├── {sample_A}.collapsed.filtered.rep.fq
│ ├── {sample_A}.collapsed.filtered.other_files
│ ├── {sample_A}.collapsed.rep.fq
│ ├── {sample_A}.collapsed.other_files
│ ├── {sample_B}.collapsed.abundance.txt
│ ├── {sample_B}.collapsed.fasta
│ ├── {sample_B}.collapsed.filtered.abundance.txt
│ ├── {sample_B}.collapsed.filtered.rep.fasta
│ ├── {sample_B}.collapsed.filtered.rep.fq
│ ├── {sample_B}.collapsed.filtered.other_files
│ ├── {sample_B}.collapsed.rep.fq
│ ├── {sample_B}.collapsed.other_files
│ ├── merged.collapsed.abundance.txt
│ ├── merged.collapsed.fasta
│ ├── merged.collapsed.filtered.abundance.txt
│ ├── merged.collapsed.filtered.rep.fasta
│ ├── merged.collapsed.filtered.rep.fq
│ ├── merged.collapsed.filtered.other_files
│ ├── merged.collapsed.rep.fq
│ └── merged.collapsed.other_files
├── kallisto_index
│ ├── complete.txt
│ └── index
├── polished # clustered polished reads
│ ├── {sample_A}.polished.bam
│ ├── {sample_A}.polished.bam.pbi
│ ├── {sample_A}.polished.cluster_report.csv
│ ├── {sample_A}.polished.hq.fasta
│ ├── {sample_A}.polished.hq.fastq
│ ├── {sample_A}.polished.hq.fastq.gz
│ ├── {sample_A}.polished.other_files
│ ├── {sample_B}.polished.bam
│ ├── {sample_B}.polished.bam.pbi
│ ├── {sample_B}.polished.cluster_report.csv
│ ├── {sample_B}.polished.hq.fasta
│ ├── {sample_B}.polished.hq.fastq
│ ├── {sample_B}.polished.other_files
│ ├── merged.polished.bam
│ ├── merged.polished.bam.pbi
│ ├── merged.polished.cluster_report.csv
│ ├── merged.polished.hq.fasta
│ ├── merged.polished.hq.fastq
│ └── merged.polished.other_files
├── sqanti # splicing analysis with non-collpased reads
│ ├── GMST
│ ├── {sample_A}.polished.hq_classification.txt
│ ├── {sample_A}.polished.hq_corrected.fasta
│ ├── {sample_A}.polished.hq_corrected.gtf
│ ├── {sample_A}.polished.hq_corrected_sorted.sam
│ ├── {sample_A}.polished.hq_junctions.txt
│ ├── {sample_A}.polished.hq_sqanti_report.pdf
│ ├── {sample_A}.polished.hq_other_files
│ ├── {sample_B}.polished.hq_classification.txt
│ ├── {sample_B}.polished.hq_corrected.fasta
│ ├── {sample_B}.polished.hq_corrected.gtf
│ ├── {sample_B}.polished.hq_corrected_sorted.sam
│ ├── {sample_B}.polished.hq_junctions.txt
│ ├── {sample_B}.polished.hq_sqanti_report.pdf
│ ├── {sample_B}.polished.hq_other_files
│ ├── merged.polished.hq_classification.txt
│ ├── merged.polished.hq_corrected.fasta
│ ├── merged.polished.hq_corrected.gtf
│ ├── merged.polished.hq_corrected.sorted.sam
│ ├── merged.polished.hq_junctions.txt
│ ├── merged.polished.hq_sqanti_report.pdf
│ ├── merged.polished.hq_other_files
│ └── other_files
├── sqanti_r2 # splicing analysis with collapsed
│ ├── GMST
│ ├── {sample_A}.collapsed.filtered.rep_classification.filtered_lite_classification.txt
│ ├── {sample_A}.collapsed.filtered.rep_classification.filtered_lite.fasta
│ ├── {sample_A}.collapsed.filtered.rep_classification.filtered_lite.gtf
│ ├── {sample_A}.collapsed.filtered.rep_classification.filtered_lite_junctions.txt
│ ├── {sample_A}.collapsed.filtered.rep_classification.filtered_lite_reasons.txt
│ ├── {sample_A}.collapsed.filtered.rep_classification.filtered_lite_sqanti_report.pdf
│ ├── {sample_A}.collapsed.filtered.rep_classification.txt
│ ├── {sample_A}.collapsed.filtered.rep_corrected.fasta
│ ├── {sample_A}.collapsed.filtered.rep_corrected.gtf
│ ├── {sample_A}.collapsed.filtered.rep_corrected_sorted.sam
│ ├── {sample_A}.collapsed.filtered.rep_junctions.txt
│ ├── {sample_A}.collapsed.filtered.rep_sqanti_report.pdf
│ ├── {sample_A}.collapsed.filtered.rep_other_files
│ ├── {sample_B}.collapsed.filtered.rep_classification.filtered_lite_classification.txt
│ ├── {sample_B}.collapsed.filtered.rep_classification.filtered_lite.fasta
│ ├── {sample_B}.collapsed.filtered.rep_classification.filtered_lite.gtf
│ ├── {sample_B}.collapsed.filtered.rep_classification.filtered_lite_junctions.txt
│ ├── {sample_B}.collapsed.filtered.rep_classification.filtered_lite_reasons.txt
│ ├── {sample_B}.collapsed.filtered.rep_classification.filtered_lite_sqanti_report.pdf
│ ├── {sample_B}.collapsed.filtered.rep_classification.txt
│ ├── {sample_B}.collapsed.filtered.rep_corrected.fasta
│ ├── {sample_B}.collapsed.filtered.rep_corrected.gtf
│ ├── {sample_B}.collapsed.filtered.rep_corrected_sorted.sam
│ ├── {sample_B}.collapsed.filtered.rep_junctions.txt
│ ├── {sample_B}.collapsed.filtered.rep_sqanti_report.pdf
│ ├── {sample_B}.collapsed.filtered.rep_other_files
│ ├── merged.collapsed.filtered.rep_classification.txt
│ ├── merged.collapsed.filtered.rep_corrected.fasta
│ ├── merged.collapsed.filtered.rep_corrected.gtf
│ ├── merged.collapsed.filtered.rep_corrected.sorted.sam
│ ├── merged.collapsed.filtered.rep_junctions.txt
│ ├── merged.collapsed.filtered.rep_sqanti_report.pdf
│ ├── merged.collapsed.filtered.rep_tappAS_annot_from_SQANTI3.gff3
│ ├── merged.collapsed.filtered.rep_other_files
│ └── other_files
├── subread
│ ├── {sample_A}.bam
│ ├── {sample_A}.bam.pbi
│ ├── {sample_A}.subreadset.xml
│ ├── {sample_B}.bam
│ ├── {sample_B}.bam.pbi
│ ├── {sample_B}.subreadset.xml
│ ├── merged.bam
│ └── merged.bam.pbi
└── targeted # targeted non-collapsed reads
├── {sample_A}.polished.hq_corrected_sorted_targeted.sam
├── {sample_B}.polished.hq_corrected_sorted_targeted.sam
└── merged.polished.hq_corrected.sorted.targeted.sam