Longbow """""""

|GitHub release| |Generic badge| |PyPI version maslongbow|

.. |GitHub release| image:: https://img.shields.io/github/release/broadinstitute/longbow.svg :target: https://github.com/broadinstitute/longbow/releases/

.. |Generic badge| image:: https://img.shields.io/badge/Docker-v0.6.14-blue.svg :target: https://console.cloud.google.com/gcr/images/broad-dsp-lrma/US/lr-longbow

.. |PyPI version maslongbow| image:: https://img.shields.io/pypi/v/maslongbow.svg :target: https://pypi.python.org/pypi/maslongbow/

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 <https://broadinstitute.github.io/longbow/>_.

Installation

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/

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 <https://broadinstitute.github.io/longbow/commands.html>_.

::

# 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

Getting help

The Longbow documentation page <https://broadinstitute.github.io/longbow/> 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 <https://github.com/broadinstitute/longbow/issues>.

Developers' guide

For information on contributing to Longbow development, visit our developer documentation <DEVELOP.md>_.