SMART-3SEQ SNAKEMAKE WORKFLOW v1.0

Snakemake workflow, environment and analysis file structure for sequencing data produced using SMART-3Seq method. The SMART-3Seq method is designed for RNA-seq from archival tissue, as well as from laser-capture microdissected tissue, created by J. Foley et al.

Installation

Clone this repository:

git clone https://github.com/danielanach/SMART-3SEQ-smk.git
cd SMART-3SEQ-smk

This project uses conda as an environment manager.

Create an environment and install dependencies:

conda env create -f environment.yml

Enter environment:

conda activate smart-3seq

Clone 3SEQtools repositories for UMI-handling tools.

cd code/

git clone https://github.com/jwfoley/3SEQtools.git

git clone https://github.com/jwfoley/umi-dedup.git

Obtain required reference files

You can download GTF files and FASTA files from here.

You need the reference genome indexed for the STAR aligner. There are some pre-made STAR indexed genomes here. I’d recommend one under the “GENCODE” directory so that there is gene annotation.

If you'd like to generate your own, follow the instructions in the STAR manual under "2 Generating genome indexes.”

Configure snakemake yaml

Edit config.yaml for your sequencing run specific details.

# Sample information
SAMPLES:
  - "Sample_1"
  - "Sample_2"
  - "Sample_3"
  - "Sample_4"

# Read group names to use in bam files
RG_NAME: "PROJECT_NAME"

# Adapter sequences
ADAPTER_3_PRIME: "AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC"
ADAPTER_5_PRIME: "ATCTCGTATGCCGTCTTCTGCTTG"

# 3SEQ-tools parameters
N: "5"  # Length of UMI
G: "3"  # Length of G-overhang
A: "8"  # Minimum length of homopolymer detection
M: "1"  # Number of mismatches allowed

# Other parameters
ML: "5" # Minimum length read after adapter trimming

# Reference info
REF_GENOME: "/path/genome.fa"
GTF: "/path/annotation.gtf"
STAR_DIR: "/path/star_directory"
TEMP_DIR: "/path/_STARtmp"

# Software
THREE_SEQ: "./3SEQtools"
UMI_TOOLS: "./umi-dedup"

# CPU information
RAM: "2000000000"

# Project directory, must exist
PROJECT_DIR: "/path/output"

# Fastq directory which contains fastq.gz files corresponding to SAMPLES
FASTQ_DIR: "/scratch/dana/3seq_DCIS_reg/fastq"

Execute pipeline

snakemake --snakefile pipeline.smk --configfile config.yaml -j #_of_cores

File structure post-analysis

└── PROJECT_DIR/
│── trimmed
│ │── sample.R1.trimmed.fastq (adapter trimmed)
│ └── sample.R1.trimmed_umi.fastq (UMI polyAtrimmed)
│── bam
│ │── sample.bam
│ └── sample.bam.bai
│── dedup_bam
│ │── sample.dedup.bam
│ └── sample.dedup.bam.bai
│── logs
│ │── sampleLog.final.out (STAR log)
│ │── sampleLog.progress.out (STAR log)
│ │── sampleLog.std.out (STAR log)
│ │── sampleLog.out.tab (STAR log)
│ └── star_remove_genome.log (STAR log)
└── counts
│── counts.txt
└── counts.txt.summary