maslongbow 0.4.5.8

Annotation and segmentation of MAS-seq data

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.

Installation

pip is recommended for Longbow installation.

pip install maslongbow

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/

Getting Started

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

# Annotate reads according to the mas15 model
longbow annotate -m mas15 -o ann.bam mas15_test_input.bam

# Generate annotation stats and automatic QC figures in .png and .svg format
longbow stats -o stats ann.bam

# Segment reads according to the model annotations
longbow segment -o seg.bam ann.bam

# Filter out improperly-constructed arrays (will generate filter_passed.bam and filter_failed.bam files)
longbow filter -o filter seg.bam

# 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

Getting help

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.

Developers’ guide

For information on contributing to Longbow development, visit our developer documentation.