Genetic multiplexing of barcoded single cell RNA-seq
Demuxlet has been published: https://www.nature.com/articles/nbt.4042
If you find it useful, please cite: Kang et al., Nature Biotechnology 2017.
--alpha 0 --alpha 0.5, which assumes the expected proportion of 50% genetic mixture from two individuals, to get better estimates of doublets.--group-list to a list of barcodes (i.e. barcodes.tsv from 10X) to speed things up and only get demultiplexing for cells called by other methodsdemuxlet is a software tool to deconvolute sample identity and identify multiplets when multiple samples are pooled by barcoded single cell sequencing. demuxlet takes (1) a SAM/BAM/CRAM file produced by the standard 10x sequencing platform, or any other barcoded single cell RNA-seq (with proper --tag-UMI and --tag-group) options (2) a VCF/BCF file containing the genotype (GT), posterior probability (GP), or genotype likelihood (GL) to assign each barcode to a specific sample (or a pair of samples) in the VCF file.
Before installing demuxlet, you need to install htslib in the same directory you want to install demuxlet (i.e. demuxlet and htslib should be siblings).
NOTE htslib 1.11 is not supported for now - use earlier releases (e.g. 1.10.x)
After installing htslib, you can clone the current snapshot of this repository to install as well
$ git clone https://github.com/statgen/demuxlet.git $ cd demuxlet $ autoreconf -vfi $ ./configure (with additional options such as --prefix) $ make $ make install (may require root privilege)
demuxlet uses a self-documentation utility. You can run each utility with -man or -help option to see the command line usages.
$ ./demuxlet (for short usage) $ ./demuxlet -help (for detailed usage)
The detailed usage is also pasted below.
Options for input SAM/BAM/CRAM --sam [STR: ] : Input SAM/BAM/CRAM file. Must be sorted by coordinates and indexed --tag-group [STR: CB] : Tag representing readgroup or cell barcodes, in the case to partition the BAM file into multiple groups. For 10x genomics, use CB --tag-UMI [STR: UB] : Tag representing UMIs. For 10x genomiucs, use UB Options for input VCF/BCF --vcf [STR: ] : Input VCF/BCF file, containing the individual genotypes (GT), posterior probability (GP), or genotype likelihood (PL) --field [STR: GP] : FORMAT field to extract the genotype, likelihood, or posterior from --geno-error [FLT: 0.01] : Genotype error rate (must be used with --field GT) --min-mac [INT: 1] : Minimum minor allele frequency --min-callrate [FLT: 0.50] : Minimum call rate --sm [V_STR: ] : List of sample IDs to compare to (default: use all) --sm-list [STR: ] : File containing the list of sample IDs to compare Output Options --out [STR: ] : Output file prefix --alpha [V_FLT: ] : Grid of alpha to search for (default is 0.1, 0.2, 0.3, 0.4, 0.5) --write-pair [FLG: OFF] : Writing the (HUGE) pair file --doublet-prior [FLT: 0.50] : Prior of doublet --sam-verbose [INT: 1000000] : Verbose message frequency for SAM/BAM/CRAM --vcf-verbose [INT: 10000] : Verbose message frequency for VCF/BCF Read filtering Options --cap-BQ [INT: 40] : Maximum base quality (higher BQ will be capped) --min-BQ [INT: 13] : Minimum base quality to consider (lower BQ will be skipped) --min-MQ [INT: 20] : Minimum mapping quality to consider (lower MQ will be ignored) --min-TD [INT: 0] : Minimum distance to the tail (lower will be ignored) --excl-flag [INT: 3844] : SAM/BAM FLAGs to be excluded Cell/droplet filtering options --group-list [STR: ] : List of tag readgroup/cell barcode to consider in this run. All other barcodes will be ignored. This is useful for parallelized run --min-total [INT: 0] : Minimum number of total reads for a droplet/cell to be considered --min-uniq [INT: 0] : Minimum number of unique reads (determined by UMI/SNP pair) for a droplet/cell to be considered --min-snp [INT: 0] : Minimum number of SNPs with coverage for a droplet/cell to be considered
demuxlet generates multiple output file, such as [prefix].best, [prefix].sing, [prefix].sing2, and optionally [prefix].pair (with --write-pair argument). Each file contains the following information
[prefix].best file contains the best guess of the sample identity, with detailed statistics to reach to the best guess[prefix].sing file contains the statistics for matching each cell with each possible sample.[prefix].sing2 file contains the statistics similar information to the previous one, but generated for sanity checking of the [prefix].pair results.[prefix].pair file contains the statistics for matching each cell with each possible configuration of doublet. The [prefix].best file contains the following 22 columns.