HongYhong / freddie

Freddie: Annotation-independent detection and discovery of transcriptomic alternative splicing isoforms using long-read sequencing

Freddie

Running Freddie using Snakemake

The whole Freddie pipeline is readily available using Snakemake. You can check the pipeile at the Snakefile and you can add samples and other paths (e.g. Gurobi licence, reference genome) to run Freddie on in the config.yaml file. After editing config.yaml, you can run Snakemake with your specific settings, just make sure to use --use-conda to have all the requirements installed on the fly. Note that the cluster stage uses Gurobi solver which needs a license to use. If your affliation is academic, you can cost-free obtain a license here. Make sure to update the license path in config.yaml to point to the installed license file.

Running Freddie manually

Installation

The simplest way to install the dependencies is using Conda:

git clone https://github.com/vpc-ccg/freddie.git
cd freddie
conda env create -f envs/freddie.yml
conda activate freddie

There are few scripts/stages in Freddie:

• py/freddie_split.py: Partitions the reads into independent sets that can be processed in parallel
• py/freddie_segment.py: Computes the canonical segmentation for each read set
• py/freddie_cluster.py: Clusters the reads using their canonical segmentation representation
• py/freddie_isoforms.py: Generates consensus isoforms of each cluster and outputs them in GTF format

Align

minimap2 -a -x splice -t {threads} {genome FASTA} {reads FASTA/FASTQ} > {SAM}

Sort

Before running split stage, the SAM file needs to be sorted and indexed using SAMtools

samtools sort {SAM} -m {memory per thread e.g. 2GB} -@ {threads} -O bam > {BAM}
samtools index {BAM}

Split (aka partition)

py/freddie_split.py --reads {reads FASTQ} --bam {sorted BAM} --outdir {SPLIT} -t {threads}

Align takes the following arguments:

• --reads/-r: Space separated paths to reads in FASTQ or FASTA
• --bam/-b: BAM file of read alignments from a split/splice long-read mapper that are position sorted and indexed.
• --outdir/-o: Output TSV file of split stage. Default: freddie_split/

Segment

py/freddie_segment.py -s {SPLIT} --outdir  {SEGMENT} -t {threads}

Align takes the following arguments:

• --split-dir/-s: SPLIT output directory of the split stage
• --threads/-t: Number of threads. Default: 1
• --sigma/-sd: Standard deviation parameter for the Gaussian filter. Default: 5.0
• --threshold-rate/-tp: Coverage percentage threshold for segments. Default: 0.90
• --variance-factor/-vf: Variance factor for heuristic of prefixing breakpoints. Any breakpoint with signal greater than -vf times the standard deviation plus the average of the signals will be prefixed. Default: 3.0
• --max-problem-size/-mps: Maximum allowed problem size after which the problem will be broken down uniformly. Default: 50
• --min-read-support-outside: Minimum contrasting coverage support required for a breakpoint. Default: 3
• --outdir/-o: Output directory of segment stage. Default: freddie_segment/

Cluster

The cluster stage uses Gurobi solver which needs a license to use. If your affliation is academic, you can cost-free obtain a license here.

export GRB_LICENSE_FILE={path to Gurobi v9 license}
py/freddie_cluster.py --segment-dir {SEGMENT} --outdir {CLUSTER}

Align takes the following arguments:

• --segment-dir/-s: SEGMENT output directory of the segment stage
• --gap-offset/-go: Constant +/- slack used in unaligned gap condition. Default: 20
• --epsilon/-e: +/- ratio of length as slack used in unaligned gap condition. Default: 0.2
• --max-rounds/-mr: Maximum allowed number of rounds per sub-partition of a read set. Default: 30
• --min-isoform-size/-is: Minimum read support allowed for an isoform. Default: 3
• --timeout/-to: Gurobi timeout per isoform in minutes. Default: 4
• --threads/-t: Number of threads. Default: 1
• --logs-dir/-l: Directory path where logs will be outputted. Default: No logging
• --outdir/-o: Output directory of cluster stage. Default: freddie_cluster/

Isoforms

py/freddie_isoforms.py --split-dir {SPLIT} --cluster-dir {CLUSTER} --output {ISOFORMS.GTF} -t {threads}

Align takes the following arguments:

• --split-dir/-s: SPLIT output directory of the segment stage
• --cluster-dir/-c: CLUSTER output directory of the cluster stage
• --output/-o: Output GTF file of isoforms stage. Default: freddie_isoforms.gtf
• --threads/-t: Number of threads. Default: 1