HCMV RNA-seq Analysis Pipeline

Overview

This repository contains a reproducible Snakemake-based Python pipeline for analyzing Human cytomegalovirus (HCMV; Human cytomegalovirus) transcriptomes from RNA-seq data.

Overview of Analysis

We compared HCMV transcriptomes from;

Donor	Timepoint	SRA Accession
Donor 1	2 dpi	SRR5660030
Donor 1	6 dpi	SRR5660033
Donor 3	2 dpi	SRR5660044
Donor 3	6 dpi	SRR5660045

All the results are summarized in a single output file called Ofosu_PipelineReport.txt

What the pipeline does

For each sample, the pipeline performs the following steps:

---

• Download SRA reads and converted to fastq (Documented only)
• Extracts coding sequences (CDS) sequences from the HCMV reference genome
• Builds a kallisto transcriptome index
• Quantifies transcript expression using kallisto
• Runs sleuth differential expression analysis (R script)
• Builds a Bowtie2 genome index
• Maps reads to the HCMV genome
• Counts reads before and after mapping
• Assembles RNA reads using SPAdes
• Extracts the longest contig from each assembly
• Runs BLASTN of the longest contig against a Betaherpesvirinae nucleotide database
• Generate a PipelineReport.txt

---

Download SRA Data

# Example for one sample
fasterq-dump SRR5660030 --split-files

Repeated for :

• SRR5660030

• SRR5660033

• SRR5660044

• SRR5660045

These files were subsampled into TESTS/ used in the pipeline

Dependencies

The following tools and softwares must be installed and available:

• snakemake 7.32.4 (https://snakemake.readthedocs.io/en/v7.32.0/getting_started/installation.html)
• Python 3.12.3 (https://www.python.org/downloads/release/python-3123/)
• R 4.5.2 (https://cran.r-project.org/bin/windows/base/)
• kallisto 0.51.1 (https://pachterlab.github.io/kallisto/download)
• bowtie2 2.5.2 (https://anaconda.org/bioconda/bowtie2)
• sleuth (https://pachterlab.github.io/sleuth/download)
• spades v3.15.5 (https://github.com/ablab/spades/releases)
• conda (https://anaconda.org/anaconda/conda)

Downloading the repository

In your Terminal, run in any directory of your choice:

git clone https://github.com/bofosu01/rna_seq_analysis.git

Moving into the directory containing all the files in the github repository

cd rna_seq_analysis

total 24K
drwxr-xr-x 2 bofosu student 4.0K Feb 22 19:15 BLAST

drwxr-xr-x 2 bofosu student 4.0K Feb 22 19:15 GCF_000845245.1

-rw-r--r-- 1 bofosu student 2.2K Feb 22 19:15 README.md

drwxr-xr-x 2 bofosu student 4.0K Feb 22 19:15 SCRIPTS

drwxr-xr-x 3 bofosu student 4.0K Feb 22 19:16 SNAKEMAKE

drwxr-xr-x 2 bofosu student 4.0K Feb 22 19:15 TESTS

Move into the SNAKEMAKE directory. It contains the Snakefile

cd SNAKEMAKE

total 8.0K
-rw-r--r-- 1 bofosu student 7.7K Feb 22 19:15 Snakefile

How to run the pipeline

Dry run Check that everything is connected correctly

snakemake --dry-run

Run the pipeline

snakemake --cores 4

Results

total 32K
drwxr-xr-x  2 bofosu student 4.0K Feb 22 19:15 BLAST

drwxr-xr-x  2 bofosu student 4.0K Feb 22 19:15 GCF_000845245.1

-rw-r--r--  1 bofosu student 2.6K Feb 22 19:25 PipelineReport.txt

drwxr-xr-x 11 bofosu student 4.0K Feb 22 19:25 OUTPUTS

-rw-r--r--  1 bofosu student 2.2K Feb 22 19:15 README.md

drwxr-xr-x  2 bofosu student 4.0K Feb 22 19:25 SCRIPTS

drwxr-xr-x  3 bofosu student 4.0K Feb 22 19:25 SNAKEMAKE

drwxr-xr-x  2 bofosu student 4.0K Feb 22 19:15 TESTS