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Quick Start Guide

Get started with dnbc4tools

◆ RNA-seq • ◆ ATAC-seq • ◆ VDJ-seq

Prerequisites

Before you begin:

◆ Single-Cell RNA Analysis

Gene expression profiling at single-cell resolution

Step 1: Build Reference Genome

Human (GRCh38)

# Download reference files
wget http://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_32/GRCh38.primary_assembly.genome.fa.gz
wget http://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_32/gencode.v32.primary_assembly.annotation.gtf.gz

# Extract files
gzip -d GRCh38.primary_assembly.genome.fa.gz
gzip -d gencode.v32.primary_assembly.annotation.gtf.gz

# Build reference
$dnbc4tools tools mkgtf --ingtf gencode.v32.primary_assembly.annotation.gtf --output genes.filter.gtf --type gene_type
$dnbc4tools rna mkref --ingtf genes.filter.gtf --fasta GRCh38.primary_assembly.genome.fa --threads 10 --species Homo_sapiens

Mouse (GRCm38)

# Download reference files
wget http://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M23/GRCm38.primary_assembly.genome.fa.gz
wget http://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M23/gencode.vM23.primary_assembly.annotation.gtf.gz

# Extract files
gzip -d GRCm38.primary_assembly.genome.fa.gz
gzip -d gencode.vM23.primary_assembly.annotation.gtf.gz

# Build reference
$dnbc4tools tools mkgtf --ingtf gencode.vM23.primary_assembly.annotation.gtf --output genes.filter.gtf --type gene_type
$dnbc4tools rna mkref --ingtf genes.filter.gtf --fasta GRCm38.primary_assembly.genome.fa --threads 10 --species Mus_musculus

Human-Mouse Mixed Reference

# Prepare both references as above, then:
$dnbc4tools rna mkref \
    --fasta GRCh38.primary_assembly.genome.fa,GRCm38.primary_assembly.genome.fa \
    --ingtf hg38/genes.filter.gtf,mm10/genes.filter.gtf \
    --species hg38,mm10 \
    --threads 10

Step 2: Run Analysis

Standard RNA-seq Analysis

$dnbc4tools rna run \
    --cDNAfastq1 /test/data/test_cDNA_R1.fastq.gz \
    --cDNAfastq2 /test/data/test_cDNA_R2.fastq.gz \
    --oligofastq1 /test/data/test_oligo1_1.fq.gz,/test/data/test_oligo2_1.fq.gz \
    --oligofastq2 /test/data/test_oligo1_2.fq.gz,/test/data/test_oligo2_2.fq.gz \
    --genomeDir /database/scRNA/Mus_musculus/mm10 \
    --name test \
    --threads 30

◆ Single-Cell ATAC Analysis

Chromatin accessibility profiling at single-cell resolution

Step 1: Build Reference Genome

Human (GRCh38)

# Download reference files
wget http://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_32/GRCh38.primary_assembly.genome.fa.gz
wget http://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_32/gencode.v32.primary_assembly.annotation.gtf.gz

# Extract files
gzip -d GRCh38.primary_assembly.genome.fa.gz
gzip -d gencode.v32.primary_assembly.annotation.gtf.gz

# Build reference
$dnbc4tools tools mkgtf --ingtf gencode.v32.primary_assembly.annotation.gtf --output genes.filter.gtf --type gene_type
$dnbc4tools atac mkref --fasta GRCh38.primary_assembly.genome.fa --ingtf genes.filter.gtf --species Homo_sapiens --prefix chr

Mouse (GRCm38)

# Download reference files
wget http://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M23/GRCm38.primary_assembly.genome.fa.gz
wget http://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M23/gencode.vM23.primary_assembly.annotation.gtf.gz

# Extract files
gzip -d GRCm38.primary_assembly.genome.fa.gz
gzip -d gencode.vM23.primary_assembly.annotation.gtf.gz

# Build reference
$dnbc4tools tools mkgtf --ingtf gencode.vM23.primary_assembly.annotation.gtf --output genes.filter.gtf --type gene_type
$dnbc4tools atac mkref --fasta GRCm38.primary_assembly.genome.fa --ingtf genes.filter.gtf --species Mus_musculus --prefix chr

Step 2: Run Analysis

ATAC-seq Analysis

$dnbc4tools atac run \
    --fastq1 /test/data/test1_R1.fastq.gz,/test/data/test2_R1.fastq.gz \
    --fastq2 /test/data/test1_R2.fastq.gz,/test/data/test2_R2.fastq.gz \
    --genomeDir /database/scATAC/Mus_musculus/mm10 \
    --name test \
    --threads 10

◆ Single-Cell VDJ Analysis

Immune receptor repertoire profiling (requires 5' RNA-seq data)

⚠️ **Prerequisite**: Complete 5' scRNA analysis first to establish cell-bead correspondence.

Step 1: 5' RNA Analysis

5' scRNA-seq Analysis

$dnbc4tools rna run \
    --cDNAfastq1 /test/data/test_cDNA_R1.fastq.gz \
    --cDNAfastq2 /test/data/test_cDNA_R2.fastq.gz \
    --oligofastq1 /test/data/test_oligo1_1.fq.gz,/test/data/test_oligo2_1.fq.gz \
    --oligofastq2 /test/data/test_oligo1_2.fq.gz,/test/data/test_oligo2_2.fq.gz \
    --genomeDir /database/scRNA/Homo_sapiens \
    --name test \
    --threads 30 \
    --end5

Step 2: VDJ Analysis

TCR Analysis (Human)

$dnbc4tools vdj run \
    --fastq1 /test/data/test1_R1.fastq.gz,/test/data/test2_R1.fastq.gz \
    --fastq2 /test/data/test1_R2.fastq.gz,/test/data/test2_R2.fastq.gz \
    --beadstrans /scRNA/test1/outs/singlecell.csv \
    --ref human \
    --name test_human_tcr \
    --threads 20 \
    --chain TR

TCR Analysis (Mouse)

$dnbc4tools vdj run \
    --fastq1 /test/data/test3_R1.fastq.gz,/test/data/test4_R1.fastq.gz \
    --fastq2 /test/data/test3_R2.fastq.gz,/test/data/test4_R2.fastq.gz \
    --beadstrans /scRNA/test2/outs/singlecell.csv \
    --ref mouse \
    --name test_mouse_tcr \
    --threads 20 \
    --chain TR

BCR Analysis (Human)

$dnbc4tools vdj run \
    --fastq1 /test/data/test5_R1.fastq.gz,/test/data/test6_R1.fastq.gz \
    --fastq2 /test/data/test5_R2.fastq.gz,/test/data/test6_R2.fastq.gz \
    --beadstrans /scRNA/test1/outs/singlecell.csv \
    --ref human \
    --name test_human_bcr \
    --threads 20 \
    --chain IG

BCR Analysis (Mouse)

$dnbc4tools vdj run \
    --fastq1 /test/data/test7_R1.fastq.gz,/test/data/test8_R1.fastq.gz \
    --fastq2 /test/data/test7_R2.fastq.gz,/test/data/test8_R2.fastq.gz \
    --beadstrans /scRNA/test2/outs/singlecell.csv \
    --ref mouse \
    --name test_mouse_bcr \
    --threads 20 \
    --chain IG

◆ Command Reference

Essential Commands Summary

Workflow Command Purpose
RNA Analysis dnbc4tools rna run Complete RNA-seq pipeline
ATAC Analysis dnbc4tools atac run Complete ATAC-seq pipeline
VDJ Analysis dnbc4tools vdj run TCR/BCR repertoire analysis
Reference Building dnbc4tools rna mkref Build RNA reference database
Reference Building dnbc4tools atac mkref Build ATAC reference database
GTF Processing dnbc4tools tools mkgtf Filter and process GTF files

Further Reading