mutCaller

Pipeline for extracting variants from single cell genomics data

Overview

mutCaller is a command line tool written in Rust for identifying SNVs in single cell data that contains a cell barcode (CB) and UMI such as 10X genomics data files. With mutCaller, you can supply variants (VCF file support coming), and obtain a counts file of the number of variants that map to your query and their associated cell-barcodes and umis. mutCaller using the takes as input either unaligned fastqs (UNALIGNED function) or an aligned BAM file with the CB and UMI as tags (ALIGNED function).

Installation

mutCaller is written in Rust. To install the rust compiler go to https://www.rust-lang.org/tools/install. mutCaller requires two additional tools be available on the command line, minimap2 (https://github.com/lh3/minimap2) and samtools (https://samtools.github.io).

To install mutCaller:

clone the repository by typing https://github.com/furlan-lab/mutCaller.git from the location you want to build from
enter the cloned repo by typing cd mutCaller
build by typing cargo build --release
the build process will create a self contained binary executable file in targets/release directory called mutCaller
move this binary elsewhere if desired (ideally somewhere referenced by your PATH environment variable - e.g. ~/.local/bin)

Updates

version 0.40 - 6/14/23 - majorcode cleanup, added STAR aligner option, added vcf file support including a VARIANT subfunction that allows you to troubleshoot variants files version 0.30 - 5/9/23 - code cleanup and implemented bam option version 0.22 - 5/9/23 - first alpha release

Usage

Variants file (sser generated)

Users can generate a simple, 'variants_file' that lists the variants to be counted (tsv.file). The variants file should look like this:

seqname\tstart\tref_nt\tquery_nt\tname

More detailed explanation:

seqname - e.g. 'chr1', 'chr2', etc
position - 1-indexed position (e.g, '112450407')
ref_nt - nucleotide of the reference at this location
query_nt - nucleotide of your query
name - a string given to the name the variant in the outpull file

three full lines should look something like this

seq     start  ref_nt query_nt name
chr12   112450407   A   G   PTPN11_227A>G
chr12   208248389   G   A   IDH1_132G>A
chr17   7674220 C   T   TP53_248C>T

Barcodes file

A barcode whitelist. v3 3prime and v2 5prime 10X whitelists are provided in the data folder.

Invocation

To run mutCaller simply type:

mutCaller ALIGNED --bam <file.bam> -s <variants.tsv> -o <folder>
mutCaller UNALIGNED --barcodes_file <barcodes_file> --fastq1 <fastq1> --fastq2 <fastq2> --genome <genome> --variants <variants.tsv>

OTHER EXAMPLES

More examples on how to use mutcaller can be found here

Help menus

mutcaller 0.4.0
Scott Furlan
Single nucleotide variant counting pipeline for single cell genomics data

USAGE:
    mutcaller [SUBCOMMAND]

FLAGS:
    -h, --help       Prints help information
    -V, --version    Prints version information

SUBCOMMANDS:
    ALIGNED      Count variants in previously aligned data
    UNALIGNED    Count variants after aligning data
    VCF          variants file debugging
    help         Prints this message or the help of the given subcommand(s)

Note: this is a work in progress. Use with caution.

ALIGNED help


mutcaller-ALIGNED
Count variants in previously aligned data

USAGE:
    mutcaller ALIGNED [FLAGS] [OPTIONS] --bam <bam> --variants <variants>

FLAGS:
    -h, --help       Prints help information
    -V, --version    Prints version information
    -v, --verbose    use this flag to run in verbose mode

OPTIONS:
    -b, --bam <bam>              aligned bam file with cell barcode and umi in tags
    -c, --cb_tag <cb>            bam tag containing cell barcode; default = 'CB'
    -o, --output <output>        output path (defaults to 'out'); inside folder a counts file will be called
                                 'counts.txt.gz', log will be called mutcaller.log
    -q, --qual <qual>            filter for variant quality (float); default = 95.0; only used if VCF file type is
                                 supplied
    -t, --threads <threads>      threads
    -u, --umi_tag <umi>          bam tag containing umi; default = 'XM'
    -s, --variants <variants>    path to variants.tsv or vcf file (SNVs only supported currently); For tsv, example
                                 formating = seqname\tstart\tref_nt\tquery_nt\tname; e.g.
                                 chr12,112450407,A,G,PTPN11_227A>G

UNALIGNED help

mutcaller-UNALIGNED
Count variants after aligning data

USAGE:
    mutcaller UNALIGNED [FLAGS] [OPTIONS] --barcodes_file <barcodes_file> --fastq1 <fastq1> --fastq2 <fastq2> --genome <genome> --variants <variants>

FLAGS:
    -h, --help          Prints help information
    -k, --keep_files    use this flag to keep files (default is remove intermediate files)
    -V, --version       Prints version information
    -v, --verbose       use this flag to run in verbose mode

OPTIONS:
    -a, --aligner <aligner>                aligner software - currently minimap (default) and STAR are supported; if not
                                           available on command line supply in loc_aligner argument
    -l, --aligner_loc <aligner_loc>        path to aligner e.g. /app/software/CellRanger/6.0.1/lib/bin/STAR
    -b, --barcodes_file <barcodes_file>    barcodes_file
    -c, --cb_length <cb_len>               length of umi sequence
    -i, --fastq1 <fastq1>                  input fastq with barcodes
    -j, --fastq2 <fastq2>                  input fastq with read
    -g, --genome <genome>                  fasta for minimap2 or transcriptome index for kallisto
    -o, --output <output>                  output path (defaults to 'out'); inside folder a counts file will be called
                                           'counts.txt.gz', log will be called mutcaller.log
    -q, --qual <qual>                      filter for variant quality (float); default = 95.0; only used if VCF file
                                           type is supplied
    -r, --read_len <read_len>              read 2 length (default 90)
    -t, --threads <threads>                threads
    -u, --umi_length <umi_len>             length of umi sequence
    -s, --variants <variants>              path to variants.tsv or vcf file (SNVs only supported currently); For tsv,
                                           example formating = seqname\tstart\tref_nt\tquery_nt\tname; e.g.
                                           chr12,112450407,A,G,PTPN11_227A>G

scfurl / mutCaller