Step 2: Preparing input files for genetic demultiplexing

In Step 2, we will define the necessary files needed for Ensemblex's constituent genetic demultiplexing tools and will place them within the working directory. The necessary files vary depending on the version of the Ensemblex pipeline being used:

Demultiplexing with prior genotype information
Demultiplexing without prior genotype information

Demultiplexing with prior genotype information

Required files

To demultiplex the pooled samples with prior genotype information, the following files are required:

File	Description
gene_expression.bam	Gene expression bam file of the pooled samples (e.g., 10X Genomics possorted_genome_bam.bam)
gene_expression.bam.bai	Gene expression bam index file of the pooled samples (e.g., 10X Genomics possorted_genome_bam.bam.bai)
barcodes.tsv	Barcodes tsv file of the pooled cells (e.g., 10X Genomics barcodes.tsv)
pooled_samples.vcf	vcf file describing the genotypes of the pooled samples
genome_reference.fa	Genome reference fasta file (e.g., 10X Genomics: ~/Homo_sapiens.GRCh37/genome/10xGenomics/refdata-cellranger-GRCh37/fasta/genome.fa)
genome_reference.fa.fai	Genome reference fasta index file (e.g., 10X Genomics: ~/Homo_sapiens.GRCh37/genome/10xGenomics/refdata-cellranger-GRCh37/fasta/genome.fa.fai)
genotype_reference.vcf	Population reference vcf file (e.g., 1000 Genomes Project)

NOTE: We demonstrate how to download reference vcf and fasta files in the Tutorial section of the Ensemblex documentation.

Placing files into the Ensemblex pipeline working directory

First, define all of the required files:

BAM=/path/to/possorted_genome_bam.bam
BAM_INDEX=/path/to/possorted_genome_bam.bam.bai
BARCODES=/path/to/barcodes.tsv
SAMPLE_VCF=/path/to/pooled_samples.vcf
REFERENCE_VCF=/path/to/genotype_reference.vcf
REFERENCE_FASTA=/path/to/genome.fa
REFERENCE_FASTA_INDEX=/path/to/genome.fa.fai

Then, place the required files in the Ensemblex pipeline working directory:

## Define the path to the working directory
ensemblex_PWD=/path/to/working_directory

## Copy the files to the input_files directory in the working directory
cp $BAM  $ensemblex_PWD/input_files/pooled_bam.bam
cp $BAM_INDEX  $ensemblex_PWD/input_files/pooled_bam.bam.bai
cp $BARCODES  $ensemblex_PWD/input_files/pooled_barcodes.tsv
cp $SAMPLE_VCF  $ensemblex_PWD/input_files/pooled_samples.vcf
cp $REFERENCE_VCF  $ensemblex_PWD/input_files/reference.vcf
cp $REFERENCE_FASTA  $ensemblex_PWD/input_files/reference.fa
cp $REFERENCE_FASTA_INDEX  $ensemblex_PWD/input_files/reference.fa.fai

If the file transfer was successful, the input_files directory of the Ensemblex pipeline working directory will contain the following files:

working_directory
└── input_files
    ├── pooled_bam.bam
    ├── pooled_bam.bam.bai
    ├── pooled_barcodes.tsv
    ├── pooled_samples.vcf
    ├── reference.fa
    ├── reference.fa.fai
    └── reference.vcf

NOTE: You will notice that the names of the input files have been standardized, it is important that the input files have the corresonding name for the Ensemblex pipeline to work properly.

Upon placing the required files in the Ensemblex pipeline, we can proceed to Step 3 where we will demultiplex the pooled samples using Ensemblex's constituent genetic demultiplexing tools: Genetic demultiplexing by consituent tools

Demultiplexing without prior genotype information