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Demuxlet

Demuxlet is a genotype demultiplexing software that requires reference genotypes to be available for each individual in the pool. Therefore, if you don't have reference genotypes, you may want to demultiplex with one of the softwares that do not require reference genotype data (Freemuxlet <Freemuxlet-docs>, scSplit <scSplit-docs>, Souporcell <Souporcell-docs> or Vireo <Vireo-docs>)

Data

This is the data that you will need to have prepare to run Demuxlet:

Required

  • Reference SNP genotypes for each individual ($VCF)
    • Filter for common SNPs (> 5% minor allele frequency) and SNPs overlapping genes
    • Demuxlet is very sensitive to missing data in a vcf so please make sure you only have complete cases in your reference donor SNP genotype file
  • Genotype field in $VCF ($FIELD)
    • This is GP by default but could also be GT others
  • Barcode file ($BARCODES)
  • Bam file ($BAM)
    • Aligned single cell reads
  • Output directory ($DEMUXLET_OUTDIR)

Optional

  • A text file with the individual ids ($INDS)
    • File containing the individual ids (separated by line) as they appear in the vcf file
    • For example, this is the individual file <_download_files/Individuals.txt> for our example dataset

Run Demuxlet

First, let's assign the variables that will be used to execute each step.

Example Variable Settings

Below is an example of the variables that we can set up to be used in the command below. These are files provided as a test dataset <TestData> available in the Data Preparation Documentation <DataPrep-docs> Please replace paths with the full path to data on your system.

VCF=/path/to/TestData4PipelineFull/test_dataset.vcf
BARCODES=/path/to/TestData4PipelineFull/test_dataset/outs/filtered_gene_bc_matrices/Homo_sapiens_GRCh38p10/barcodes.tsv
BAM=/path/to/test_dataset/possorted_genome_bam.bam
DEMUXLET_OUTDIR=/path/to/output/demuxlet
FIELD='GP' ## this might also be GT depending on the fields in your vcf  
INDS=/path/to/TestData4PipelineFull/donor_list.txt ### optional

Popscle Pileup

Expected Resource Usage

~3-4h using a total of 91Gb memory when using 5 threads for the full Test Dataset <TestData> which contains ~20,982 droplets of 13 multiplexed donors,

First we will need to identify the number of reads from each allele at each SNP location.

With $INDS file

The $INDS file allows demuxlet to only consider the individual in this pool

singularity exec Demuxafy.sif popscle dsc-pileup --sam $BAM --vcf $VCF --group-list $BARCODES --out $DEMUXLET_OUTDIR/pileup --sm-list $INDS

HELP! It says my file/directory doesn't exist!

If you receive an error indicating that a file or directory doesn't exist but you are sure that it does, this is likely an issue arising from Singularity. This is easy to fix. The issue and solution are explained in detail in the Notes About Singularity Images <Singularity-docs>

Without $INDS file

This will use all the individuals in your reference SNP genotype $VCF. If your $VCF only has the individuals multiplexed in your pool, then the $INDS file is not required.

singularity exec Demuxafy.sif popscle dsc-pileup --sam $BAM --vcf $VCF --group-list $BARCODES --out $DEMUXLET_OUTDIR/pileup

HELP! It says my file/directory doesn't exist!

If you receive an error indicating that a file or directory doesn't exist but you are sure that it does, this is likely an issue arising from Singularity. This is easy to fix. The issue and solution are explained in detail in the Notes About Singularity Images <Singularity-docs>

If the pileup is successful, you will have these files in your $DEMUXLET_OUTDIR:

/path/to/output/demuxlet
├── pileup.cel.gz
├── pileup.plp.gz
├── pileup.umi.gz
└── pileup.var.gz

Additional details about outputs are available below in the Demuxlet Results and Interpretation <demuxlet-results>.

Popscle Demuxlet

Expected Resource Usage

~3min using a total of 7Gb memory when using 5 threads for the full Test Dataset <TestData> which contains ~20,982 droplets of 13 multiplexed donors,

Once you have run popscle pileup, you can demultiplex your samples:

With $INDS file

The $INDS file allows demuxlet to only consider the individual in this pool

singularity exec Demuxafy.sif popscle demuxlet --plp $DEMUXLET_OUTDIR/pileup --vcf $VCF --field $FIELD --group-list $BARCODES --geno-error-coeff 1.0 --geno-error-offset 0.05 --out $DEMUXLET_OUTDIR/demuxlet --sm-list $INDS

HELP! It says my file/directory doesn't exist!

If you receive an error indicating that a file or directory doesn't exist but you are sure that it does, this is likely an issue arising from Singularity. This is easy to fix. The issue and solution are explained in detail in the Notes About Singularity Images <Singularity-docs>

Without $INDS file

This will use all the individuals in your reference SNP genotype $VCF. If your $VCF only has the individuals multiplexed in your pool, then the $INDS file is not required.

singularity exec Demuxafy.sif popscle demuxlet --plp $DEMUXLET_OUTDIR/pileup --vcf $VCF --field $FIELD --group-list $BARCODES --geno-error-coeff 1.0 --geno-error-offset 0.05 --out $DEMUXLET_OUTDIR/demuxlet

HELP! It says my file/directory doesn't exist!

If you receive an error indicating that a file or directory doesn't exist but you are sure that it does, this is likely an issue arising from Singularity. This is easy to fix. The issue and solution are explained in detail in the Notes About Singularity Images <Singularity-docs>

Note

Demuxlet by default assumes that your $VCF uses R2 to indicate the imputation score. If you have a different imputation metric (INFO is also commonly used), then you should use --r2-info to indicate the metric it should use (for example: --r2-info INFO)

If demuxlet is successful, you will have these new files in your $DEMUXLET_OUTDIR:

/path/to/output/demuxlet
├── demuxlet.best
├── pileup.cel.gz
├── pileup.plp.gz
├── pileup.umi.gz
└── pileup.var.gz

Additional details about outputs are available below in the Demuxlet Results and Interpretation <demuxlet-results>.

Demuxlet Summary

We have provided a script that will summarize the number of droplets classified as doublets, ambiguous and assigned to each donor by Demuxlet and write it to the $DEMUXLET_OUTDIR. You can run this to get a fast and easy summary of your results by providing the path to your result file:

singularity exec Demuxafy.sif bash Demuxlet_summary.sh $DEMUXLET_OUTDIR/demuxlet.best

which will return:

Classification Assignment N
113_113 1334
349_350 1458
352_353 1607
39_39 1297
40_40 1078
41_41 1127
42_42 1419
43_43 1553
465_466 1094
596_597 1255
597_598 1517
632_633 868
633_634 960
660_661 1362
doublet 3053

or you can write it straight to a file:

singularity exec Demuxafy.sif bash Demuxlet_summary.sh $DEMUXLET_OUTDIR/demuxlet.best > $DEMUXLET_OUTDIR/demuxlet_summary.tsv

Note

To check if these numbers are consistent with the expected doublet rate in your dataset, you can use our Doublet Estimation Calculator.

Demuxlet Results and Interpretation

After running the Demuxlet steps and summarizing the results, you will have a number of files from some of the intermediary steps. These are the files that most users will find the most informative:

  • demuxlet.best

    • Metrics for each droplet including the singlet, doublet or ambiguous assignment (DROPLET.TYPE), final assignment (BEST.GUESS), log likelihood of the final assignment (BEST.LLK) and other QC metrics.

      INT_ID BARCODE NUM.SNPS NUM.READS DROPLET.TYPE BEST.GUESS BEST.LLK NEXT.GUESS NEXT.LLK DIFF.LLK.BEST.NEXT BEST.POSTERIOR SNG.POSTERIOR SNG.BEST.GUESS SNG.BEST.LLK SNG.NEXT.GUESS SNG.NEXT.LLK SNG.ONLY.POSTERIOR DBL.BEST.GUESS DBL.BEST.LLK DIFF.LLK.SNG.DBL
      0 AAACCTGAGATAGCAT-1

      170

      231

      SNG

      		 41_41,41_41,0.00 -29.42 40_40,41_41,0.50 -39.12 9.70 -33

      1

      		 41_41 -29.42

      597_598

      		 -76.24 0.00000 40_40,41_41,0.50 -39.12 9.70
      1 AAACCTGAGCAGCGTA-1

      325

      583

      SNG

      		 465_466,465_466,0.00 -70.61 42_42,465_466,0.50 -94.85 24.24 -74

      1

      		 465_466 -70.61

      42_42

      		 -166.61 0.00000 42_42,465_466,0.50 -94.85 24.24
      2 AAACCTGAGCGATGAC-1

      147

      227

      SNG

      		 113_113,113_113,0.00 -25.05 39_39,113_113,0.50 -29.85 4.80 -28

      1

      		 113_113 -25.05

      349_350

      		 -51.63 0.00000 39_39,113_113,0.50 -29.85 4.80
      3 AAACCTGAGCGTAGTG-1

      180

      235

      SNG

      		 349_350,349_350,0.00 -33.14 349_350,632_633,0.50 -44.78 11.64 -36

      1

      		 349_350 -33.14

      632_633

      		 -77.41 0.00000 349_350,632_633,0.50 -44.78 11.64
      4 AAACCTGAGGAGTTTA-1

      248

      444

      SNG

      		 632_633,632_633,0.00 -54.79 352_353,632_633,0.50 -72.23 17.43 -58

      1

      		 632_633 -54.79

      633_634

      		 -163.24 0.00000 352_353,632_633,0.50 -72.23 17.43
      ... ... ... ... ... ... ... ... ... ...

      ...

      		 ... ... ... ... ... ... ... ... ...

Merging Results with Other Software Results

We have provided a script that will help merge and summarize the results from multiple softwares together. See Combine Results <Combine-docs>.

Citation

If you used the Demuxafy platform for analysis, please reference our preprint as well as Demuxlet.