Analysis tools for split-seq

Requirements

Requires python 3.

Additional software needed:

• STAR
• samtools

To install all dependencies, try running install_dependencies.sh, which installs dependencies to ~/split_seq_reqs/.

To install the package: run pip install -e . (might need sudo).

Generating a reference genome

Download human reference genome

wget ftp://ftp.ensembl.org/pub/release-93/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz
gunzip Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz

Download human reference gtf file:

wget ftp://ftp.ensembl.org/pub/release-93/gtf/homo_sapiens/Homo_sapiens.GRCh38.93.gtf.gz
gunzip Homo_sapiens.GRCh38.93.gtf.gz

Download mouse reference genome

wget ftp://ftp.ensembl.org/pub/release-93/fasta/mus_musculus/dna/Mus_musculus.GRCm38.dna.primary_assembly.fa.gz
gunzip Mus_musculus.GRCm38.dna.primary_assembly.fa.gz

Download mouse reference gtf file:

wget ftp://ftp.ensembl.org/pub/release-93/gtf/mus_musculus/Mus_musculus.GRCm38.93.gtf.gz
gunzip Mus_musculus.GRCm38.93.gtf.gz

Generate split-seq reference:

split-seq mkref --genome hg38 mm10 \
                --fasta Homo_sapiens.GRCh38.dna.primary_assembly.fa Mus_musculus.GRCm38.dna.primary_assembly.fa \
                --genes Homo_sapiens.GRCh38.93.gtf Mus_musculus.GRCm38.93.gtf 
                --output_dir <ref_path>/hg38_mm10/ 
                --nthreads 16

Running the pipeline

To see all options, run split-seq -h.

split-seq all --fq1 input_R1.fastq.gz \
              --fq2 input_R2.fastq.gz \
              --output_dir <output_dir> \
              --chemistry v2 \
              --genome_dir <path_to_ref>/hg38_mm10/ \
              --nthreads 16
              --sample sample_name1 A1:B6
              --sample sample_name2 A7:B12
              --sample sample_name3 C1:D6
              --sample sample_name4 C7:D12

Merging Sublibraries into a Single Matrix

split-seq combine --output_dir <output_dir> \
                  --sublibraries <path_to_sublibrary1> <path_to_sublibrary2> ...
                  --chemistry v2
                  --genome_dir <path_to_genome_dir>
                  --sample sample_name1 <wells>

References