Longbow is a command line tool to process MAS-ISO-seq data. Longbow employs a generative modelling approach to accurately annotate and segment MAS-ISO-seq's concatenated full-length transcript isoforms from single-cell or bulk long read RNA sequencing libraries.
Documentation for all longbow commands can be found on the Longbow documentation page.
pip is recommended for Longbow installation.
pip install maslongbow
For a pre-built version including all dependencies, access our Docker image.
docker pull us.gcr.io/broad-dsp-lrma/lr-longbow:0.6.14
To install from Github source for development, the following commands can be run.
git clone https://github.com/broadinstitute/longbow.git pip install -e longbow/
The commands below illustrate the Longbow workflow on a small library of SIRVs (Spike-in RNA Variant Control Mixes). MAS-ISO-seq concatenated transcripts are annotated, segmented, and filtered using the mas15 model. A number of statistics and QC images are generated along the way. Final filtered transcripts can then be aligned using standard splice-aware long read mappers (e.g. minimap2). More detail for each command can be found in the full documentation.
# Download a tiny test dataset (less than 300K) wget https://github.com/broadinstitute/longbow/raw/main/tests/test_data/mas15_test_input.bam wget https://github.com/broadinstitute/longbow/raw/main/tests/test_data/mas15_test_input.bam.pbi wget https://github.com/broadinstitute/longbow/raw/main/tests/test_data/resources/SIRV_Library.fasta # Basic processing workflow longbow annotate -m mas_15+sc_10x5p mas15_test_input.bam | \ # Annotate reads according to the mas_15+sc_10x5p model tee ann.bam | \ # Save annotated BAM for later longbow filter | \ # Filter out improperly-constructed arrays longbow segment | \ # Segment reads according to the model longbow extract -o filter_passed.bam # Extract adapter-free cDNA sequences # Align reads with long read aligner (e.g. minimap2, pbmm2) samtools fastq filter_passed.bam | \ minimap2 -ayYL --MD -x splice:hq SIRV_Library.fasta - | \ samtools sort > align.bam && samtools index align.bam
The Longbow documentation page provides detailed descriptions of command line options and algorithmic details. If you encounter bugs or have questions/comments/concerns, please file an issue on our Github page.
For information on contributing to Longbow development, visit our developer documentation.