# download and build
git clone https://github.com/huangnengCSU/longcallR.git
cd longcallR
cargo build --release # The executable will be located at: longcallR/target/release/longcallR

# call SNPs
longcallR -b input.bam -f ref.fa -o output -t 8 -p ont-cdna       # Nanopore cDNA reads
longcallR -b input.bam -f ref.fa -o output -t 8 -p ont-drna       # Nanopore dRNA reads (no strand bias filtering)
longcallR -b input.bam -f ref.fa -o output -t 8 -p hifi-isoseq    # PacBio iso-seq reads
longcallR -b input.bam -f ref.fa -o output -t 8 -p hifi-masseq    # PacBio mas-seq reads (no strand bias filtering)

# Allele-specific junction analysis
python allele_specific/longcallR-asj.py -a annotation.gtf -b phased.bam -f ref.fa -o output_prefix -t threads

# Allele-specific expression analysis
python allele_specific/longcallR-ase.py -a annotation.gtf -b phased.bam -o output_prefix -t threads

• Introduction
• Installation
• Usage
• Demo
• Alignment
• License

LongcallR is a SNP caller for single molecule long-read RNA-seq data. LongcallR supports Nanopore cDNA sequecing and dRNA sequencing, PacBio Iso-Seq and MAS-Seq.

LongcallR is written in Rust and uses Cargo to build as follows:

git clone https://github.com/huangnengCSU/longcallR.git
cd longcallR
cargo build --release

Alternatively, install directly from crates.io:

cargo install longcallR

General usage

./longcallR \
--bam-path input.bam \                  ## The alignment bam file
--ref-path ref.fa \                     ## The reference file must be indexed.
--preset ${PRESET} \                    ## option: {ont-cdna, ont-drna, hifi-isoseq, hifi-masseq}
--output ${OUTPUT_DIR}/${PREFIX}        ## output path and prefix of output files

./longcallR -b demo/demo.bam -f demo/chr20.fa -o demo/test -t 8 -p hifi-masseq

Based on the strand orientation of reads from different PacBio and Nanopore protocols, we recommend the following alignment parameters for minimap2:

minimap2 -ax splice:hq -uf ref.fa query.fa > aln.sam    # PacBio Kinnex/Mas-seq
minimap2 -ax splice:hq ref.fa query.fa > aln.sam        # PacBio Iso-seq
minimap2 -ax splice -uf -k14 ref.fa reads.fa > aln.sam  # Nanopore dRNA
minimap2 -ax splice ref.fa reads.fa > aln.sam           # Nanopore cDNA

Note: The -uf option forces minimap2 to consider only a single transcript strand during alignment.

• PacBio Kinnex/MAS-Seq and Nanopore dRNA sequencing produce single-stranded reads.
• Nanopore cDNA sequencing produces double-stranded reads.
• PacBio Iso-Seq datasets may be either single- or double-stranded.

We recommend using the -uf option for single-stranded reads and omitting it for double-stranded reads.

MIT License

Copyright (c) 2024 Dana-Farber Cancer Institute.

Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions:

The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software.

THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.