scc-proc

Upstream processing for single cell ADT-based sequencing (including hashtags)

Necessary files

1) config.yaml file

Example configuration is shown below.

• Multiple samples can be added to the config assuming they follow the same antibody mixture and bead setup.
• All fastq files must be in the fastq_dir path
  • Specific filenames are provided in the samples attribute of the yaml file that correspond to ADT or HTO files
  • Order of fastq files in the samples attribute corresponds to the cbc, umi, tag numbering system (refer to kallisto documentation).

dirs:
  fastq_dir: "/home/user/fastq"
  out_dir: "/home/user/run/adtHto_processing/scc_proc_out"
files:
  adt_catalog: "/home/user/run/totalseqA_barcodes.csv"
  hto_catalog: "/home/user/run/totalseqAHashtag_barcodes.csv"
  allowlist: "/home/user/run/3M-february-2018.rc.txt"
general_settings:
  threads: 4
  modes: ["adt", "hto"]
  cbc: [0,0,16]
  umi: [0,16,28]
  tag: [1,0,15]
samples:
  - name: Pool_A2
  adt_fastqs: ["Pool_A2_ADT_S1_R1_001.fastq.gz", "Pool_A2_ADT_S1_R3_001.fastq.gz"]
  hto_fastqs: ["Pool_A2_HTO_S4_R1_001.fastq.gz", "Pool_A2_HTO_S4_R3_001.fastq.gz"]

2) fastq files

Refer to config above

3) Antibody file

Comma separted file with barcodes and descriptions. Example below:

CD40,CTCAGATGGAGTATG
CD44,AATCCTTCCGAATGT
CD48,CTACGACGTAGAAGA
CD21,AACCTAGTAGTTCGG

4) Allowlist cell barcode file

Allowlist file containing accepted cell barcodes. One per line. Example below:

GTCTGCTATGTCTA
GATGATGCATAGAA

Setup

1. Create conda env

conda create --name <env> --file environment_osx.txt

2. Run Snakemake

snakemake --cores 4 -s <path-to-Snakefile> --configfile=<path-to-config-yaml>

The overall process will look like this:

3. Downstream analysis of your own choosing. The count matrix and corresponding row/col info will be found in {sample_name}_{modality}_output_count/

Credits

• Conda
• Snakemake
• Kallisto
• Kallisto KITE featureMap.py
• Bustools
• kallisto | bustools KITE protocol
  • I made my Snakemake pipeline heavily based on their pipeline.