WGS Data Analysis Workflow

Step 1: Downloading Data

• Download and install SRA Toolkit

Downloading Data

Use wget to download data from SRA:

wget https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR26624132/SRR26624132

Use fastq-dump to convert SRA to fastq:

./sratoolkit.3.0.7-mac64/bin/fastq-dump --split-files SRR26624132

Step 2: Quality Control

Use FastQC to assess data quality:

fastqc SRR26624132_1.fastq SRR26624132_2.fastq

Step 3: Trimming

Use Trimmomatic to trim low-quality bases:

• Trimmomatic

java -jar trimmomatic-0.39.jar PE -phred33 SRR26624132_1.fastq SRR26624132_2.fastq trim1_paired.fastq trim1_unpaired.fastq trim2_paired.fastq trim2_unpaired.fastq ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

Step 4: Genome Assembly

Use Velvet for de novo genome assembly:

• Velvet

Install Velvet (For macOS): brew install velvet Install Velvet (For Ubuntu): sudo apt-get install velvet

velvetg ./velvet_output -clean yes -exp_cov 21 -cov_cutoff 2.81 -min_contig_lgth 200

Step 5: BLAST Analysis

Use BLAST to compare assembled contigs with a reference genome:

• blast executables Install BLAST: brew install blast or sudo apt-get install ncbi-blast+

blastp -query ./velvet_output/contigs.fa -db ref_genome -out blast_result.txt -evalue 1e-04 -outfmt 6 -max_target_seqs 5 -num_threads 8

Step 6: Functional Annotation

Retrieve top gene IDs from BLAST results:

cut -f 2 blast_result.txt | sort | uniq | head -n 10 > top_gene_ids.txt

Annotate genes using UniProt and PANTHER databases. Visit the PANTHER website and upload the gene list for functional annotation.