Pipeline for extracting variants from single cell genomics data
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 (in a convenient tsv file or by using a VCF), 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).
mutCaller is written in Rust. To install the rust compiler go to https://www.rust-lang.org/tools/install. If using the UNALIGNED function mutCaller requires additional tools be available on the command line, minimap2 (https://github.com/lh3/minimap2) or STAR (https://github.com/alexdobin/STAR) and samtools (https://samtools.github.io).
To install mutCaller:
- clone the repository by typing
https://github.com/furlan-lab/mutCaller.gitfrom 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/releasedirectory calledmutCaller - move this binary elsewhere if desired (ideally somewhere referenced by your PATH environment variable - e.g.
~/.local/bin)
- **version 0.7.0 7/24/23 - fixed a bug that caused panic in some reads that the bam library (https://gitlab.com/scfurl/bam) deemed as containing conflicts between MD tag and CIGAR string. These panics have been adressed in v 0.1.6 of the bam library and are now a required version for mutCaller
- version 0.6.* - 6/27/23 - working parallel countbam (chunking large variants into num of cores)
- version 0.5.* - 6/19/23 - indels supported, major code cleanup, checks variants.tsv file to ensure ref_nt matches the reference (for UNALIGNED-minimap2 runs only); added feature for passing additional arguments to aligner; fixed a bug that alllwed reads with different seq and qual lengths to tank STAR.
- version 0.4.* - 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
Note windows not supported; tested on MacOS and Linux (Ubuntu).
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(s) of the reference at this location
- query_nt - nucleotide(s) of your query
- name - a string given to the name the variant in the outpull file
- for all indels, the first nt provided should be contained in the reference with the following characters representing the insertion or deletion
three full lines should look something like this
Note for duplications, it is hard to predict which the aligner will detect as an insertion. We recommend for duplications that you include an insertion query assuming the first repeated sequence is detected as an insertion, then repeat for the second repeated sequence. We have given an example below
seq start ref_nt query_nt name
chr12 112450407 A G PTPN11_227A>G
chr12 112450405 T TGAG PTPN11_insT>TGAG
chr12 112450407 AGTT A PTPN11_delAGTT>A
chr6 135195908 C CGCCAGCAAGGTGCATG MYBdup1
chr6 135195925 A AGCCAGCAAGGTGCATG MYBdup2
Mutcaller will accept VCF files. Not all versions have been tested. With the VARIANTS function, a user can filter records from a VCF and optionally output a variants.tsv. It is also possible to supply a VCF to the ALIGNED and UNALIGNED functions in mutcaller directly. See examples here
A barcode whitelist. v3 3prime and v2 5prime 10X whitelists are provided in the data folder.
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>
More examples on how to use mutcaller can be found here
mutcaller 0.6.0
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
ARGPARSE test arg parsing
UNALIGNED Count variants after aligning data
VARIANTS 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.
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, sorted bam file with cell barcode and umi in tags; see cb_tag/umi_tag
-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; For tsv, example formating =
seqname\tstart\tref_nt\tquery_nt\tname; e.g. chr12,112450407,A,G,PTPN11_227A>G
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:
-x, --add_aligner_args <add_aligner_args>
additional args supplied to aligner; MUST wrap in quotes; MUST also be supplied using the long flag with
double dash and equal sign e.g. --aligner_args="--some_complex_arg parameter --some_other_arg
other_parameter", i.e. CANNOT use short flag "-x" or long flag with a space e.g. "--aligner_args
--some_complex_arg_parameter"
-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 and genome index location for STAR; for checking variants, fasta must be indexed
-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; For tsv, example formating = seqname\tstart\tref_nt\tquery_nt\tname; e.g.
chr12,112450407,A,G,PTPN11_227A>G
React
Typescript
Django
Laravel
Facebook
Microsoft
Google
Alibaba
D3
Tencent