kb-nac.smk

Snakemake workflow for UMI counting from single-nucleus RNA-seq data with kb-python, a Python wrapper for kallisto and bustools.

Note

Please make sure that you have Singularity and Snakemake installed on your system and cloned the SnakeNgs repository.

Workflow

The rulegraph was created by snakevision.

1. Build a kallisto index using the reference genome and transcriptome by kb ref with the parameter --workflow nac.
2. Quantify UMI counts using kb count with the parameter --workflow nac --h5ad --gene-names --sum total --filter bustools --overwrite.
3. Summarize and generate a report for a filtered BUS file using bustools inspect.
4. Make summary statistics using MultiQC.

Usage

snakemake -s /path/to/SnakeNgs/snakefile/kb-nac.smk \
--configfile /path/to/config.yaml \
--cores <int> \
--use-singularity \
--rerun-incomplete

config.yaml should contain the following information:

workdir: /path/to/output
experiment_table: /path/to/experiment_table.tsv
technology: 10xv3 # [10xv1, 10xv2, 10xv3, 10XV3_ULTIMA, BDWTA, INDROPSV3, Visium]
dna_fasta: /path/to/reference_dna.fa
gtf: /path/to/reference_transcriptome.gtf

• experiment_table.tsv should contain the following information:

sample  R1  R2
sample1 path/to/sample1_L001_R1.fastq.gz,path/to/sample1_L002_R1.fastq.gz path/to/sample1_L001_R2.fastq.gz,path/to/sample1_L002_R2.fastq.gz
sample2 path/to/sample2_L001_R1.fastq.gz,path/to/sample2_L002_R1.fastq.gz path/to/sample2_L001_R2.fastq.gz,path/to/sample2_L002_R2.fastq.gz
sample3 path/to/sample3_L001_R1.fastq.gz,path/to/sample3_L002_R1.fastq.gz path/to/sample3_L001_R2.fastq.gz,path/to/sample3_L002_R2.fastq.gz

R1 and R2 are comma-separated paths to the FASTQ files for read 1 and read 2, respectively.

• technology refer to the assay that generated the data. Supported assays are found in kb --list:

$ singularity exec docker://quay.io/biocontainers/kb-python:0.28.2--pyhdfd78af_2 kb --list
List of supported single-cell technologies

Positions syntax: `input file index, start position, end position`
When start & end positions are None, refers to the entire file
Custom technologies may be defined by providing a kallisto-supported technology string
(see https://pachterlab.github.io/kallisto/manual)

name            description                            on-list    barcode                    umi        cDNA
------------    -----------------------------------    -------    -----------------------    -------    -----------------------
10XV1           10x version 1                          yes        0,0,14                     1,0,10     2,None,None
10XV2           10x version 2                          yes        0,0,16                     0,16,26    1,None,None
10XV3           10x version 3                          yes        0,0,16                     0,16,28    1,None,None
10XV3_ULTIMA    10x version 3 sequenced with Ultima    yes        0,22,38                    0,38,50    0,62,None
BDWTA           BD Rhapsody                            yes        0,0,9 0,21,30 0,43,52      0,52,60    1,None,None
BULK            Bulk (single or paired)                                                                 0,None,None 1,None,None
CELSEQ          CEL-Seq                                           0,0,8                      0,8,12     1,None,None
CELSEQ2         CEL-SEQ version 2                                 0,6,12                     0,0,6      1,None,None
DROPSEQ         DropSeq                                           0,0,12                     0,12,20    1,None,None
INDROPSV1       inDrops version 1                                 0,0,11 0,30,38             0,42,48    1,None,None
INDROPSV2       inDrops version 2                                 1,0,11 1,30,38             1,42,48    0,None,None
INDROPSV3       inDrops version 3                      yes        0,0,8 1,0,8                1,8,14     2,None,None
SCRUBSEQ        SCRB-Seq                                          0,0,6                      0,6,16     1,None,None
SMARTSEQ2       Smart-seq2  (single or paired)                                                          0,None,None 1,None,None
SMARTSEQ3       Smart-seq3                                                                   0,11,19    0,11,None 1,None,None
SPLIT-SEQ       SPLiT-seq                                         1,10,18 1,48,56 1,78,86    1,0,10     0,None,None
STORMSEQ        STORM-seq                                                                    1,0,8      0,None,None 1,14,None
SURECELL        SureCell for ddSEQ                                0,0,6 0,21,27 0,42,48      0,51,59    1,None,None
Visium          10x Visium                             yes        0,0,16                     0,16,28    1,None,None

• reference_dna.fa is the reference genome in FASTA format (e.g., Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa).

• reference_transcriptome.gtf is the reference transcriptome in GTF format (e.g., Homo_sapiens.GRCh38.106.gtf).

Please refer to the tutorial for more information.

Docker image used in the workflow

• quay.io/biocontainers/kb-python:0.28.2--pyhdfd78af_2
• multiqc/multiqc:v1.28