A mitochondrial variants-based method for efficient demultiplexing of pooled single-cell RNA-seq.
git clone https://github.com/lnscan/mitoSplitter.git
conda create -n mitoSplitter python==3.9.13
conda activate mitoSplitter
pip install -r requirement.txtMake sure samtools can be used directly.
sh mitoSplitter_pipeline.sh -h ## show help informationUsage:
sh mitoSplitter_pipeline.sh <-i input.bam> <-b barcode.list> <-o out_dir> [-r bulk_matrix] [-l bulk_bam_list] [-s cor_value] [-m mito.fasta] [-t threads] [-f barcode_tag] [-q base_quality] [-a alignment_quality] [-g gold_file] [-p matrix_dir] [-d] [-h]
Description:
A mitochondrial variants-based method for efficient demultiplexing of pooled single-cell RNA-seq.
Ordering options:
-i input bam file
-b barcode list file, one barcode per line
-o name of directory for mitoSplitter output files
Other options:
-r mitochondrial variant allele frequency matrix file for all samples, must be set unless -l is set
-l bulk RNA-seq mapping bam list file for all samples, one bam file per line, must be set unless -r is set
-s warning threshold for correlation between bulk samples, used to signal samples with similar genetic backgrounds, default = 0.65
-m mitochondrial reference genome fasta, default = GRCh38_MT.fasta
-c mitochondrial reference genome id, default = MT
-t max threads to use, default = 50
-f used to extract cell barcode from bam file, default = CB
-q minimum base quality to be considered in bam file, used for variant calling, default = 20
-a minimum alignment quality to be considered in bam file, used for variant calling, default = 20
-g benchmark file used for performance validation, one barcode and the corresponding cluster per line
-p filtered feature bc matrix directory for removing doublets using scrublet, python package scrublet needs to be installed, default = FALSE
-d remove doublets using Favg Gaussian fitted model, R package mixtools needs to be installed, default = FALSE
-h print this help and exitcd example_data
sh test.sh ## result in ./example_resultIf you get this error: ../mitoSplitter_pipeline.sh: 75: [[: not found ../mitoSplitter_pipeline.sh: 78: [[: not found ERROR:Bulk mtRNA matrix path and bulk RNA-seq bam list file cannot both be missing! Please run:
cd example_data
bash test2.sh ## result in ./example_resultThank you to user hernandezvargash for providing suggestions for software installation and debugging.
Lin X, Chen Y, Lin L, Yin K, Cheng R, Lin X, Wang X, Guo Y, Wu Z, Zhang Y, Li J, Yang C, Song J. mitoSplitter: A mitochondrial variants-based method for efficient demultiplexing of pooled single-cell RNA-seq. Proc Natl Acad Sci U S A. 2023 Sep 26;120(39):e2307722120. doi: 10.1073/pnas.2307722120. Epub 2023 Sep 19. PMID: 37725654; PMCID: PMC10523499.