Hands-on Introduction to NGS Data Analysis: From Raw Sequencing Reads to Biological Interpretation

• To introduce participants to Next-Generation Sequencing and its applications in biological research.
• To explain common NGS data types and file formats such as FASTQ, FASTA, SAM/BAM, VCF, and count matrices.
• To help participants understand sequencing quality, Phred scores, read length, depth, and coverage.
• To demonstrate quality control of raw sequencing reads using commonly used tools.
• To introduce read trimming, filtering, and preprocessing workflows.
• To provide an overview of alignment, mapping, variant analysis, and RNA-Seq interpretation.
• To help participants understand how NGS outputs are interpreted for genomics and transcriptomics research.
• To guide participants in preparing a simple NGS data interpretation summary.

Day 1: Introduction to NGS and Sequencing Data Fundamentals

Focus: This session introduces participants to the fundamentals of Next-Generation Sequencing, sequencing platforms, raw sequencing reads, common file formats, and the basic structure of an NGS data analysis pipeline.

Topics Covered:

• Introduction to Next-Generation Sequencing
• Evolution from Sanger sequencing to NGS
• Major sequencing platforms: Illumina, Ion Torrent, PacBio, and Oxford Nanopore
• Applications of NGS in genomics, transcriptomics, clinical research, agriculture, microbiology, and biotechnology
• Understanding raw sequencing reads
• Introduction to NGS file formats: FASTQ, FASTA, SAM, BAM, VCF, GTF/GFF
• Understanding Phred quality score, read length, depth, and coverage
• Overview of a standard NGS data analysis pipeline
• Difference between DNA-Seq, RNA-Seq, whole genome sequencing, targeted sequencing, and metagenomics

Hands-On Activity:

• Explore sample FASTQ files
• Identify read sequence and quality score information
• Prepare a basic NGS file format interpretation sheet

Hands-On Tools:

• Sample FASTQ Dataset,Google Colab / Linux Terminal Awareness,FASTQ Viewer,Google Sheets

Day 2: Quality Control, Read Cleaning and Preprocessing

Focus: This session focuses on quality assessment of raw sequencing reads, interpretation of FastQC reports, read trimming, adapter removal, and preparation of cleaned reads for downstream analysis.

Topics Covered:

• Importance of quality control in NGS data analysis
• Introduction to raw read quality assessment
• Understanding FastQC reports
• Per-base sequence quality, GC content, sequence duplication, and adapter contamination
• Introduction to MultiQC for combined QC reporting
• Why read trimming and filtering are required
• Adapter removal and low-quality base trimming
• Overview of tools such as FastQC, MultiQC, Trimmomatic, Cutadapt, and fastp
• Comparing raw reads and cleaned reads
• Preparing a QC summary for research reporting

Hands-On Activity:

• Run or review FastQC report on sample data
• Identify quality issues in sequencing reads
• Perform/read trimming demonstration using sample data
• Prepare a short QC interpretation note

Hands-On Tools:

• FastQC,MultiQC,fastp / Trimmomatic Awareness,Google Colab

Day 3: Basic NGS Workflow Interpretation and Biological Reporting

Focus: This session introduces participants to basic downstream NGS workflow interpretation, including read alignment, mapping statistics, variant interpretation, RNA-Seq count data, differential expression awareness, visualization, and biological reporting.

Topics Covered:

• Overview of read alignment and reference genome mapping
• Introduction to aligners: BWA, Bowtie2, HISAT2, and STAR
• Understanding SAM/BAM files and mapping statistics
• Introduction to variant calling and VCF interpretation
• Introduction to RNA-Seq count data and gene expression analysis
• Basics of differential expression and biological interpretation
• Introduction to annotation and pathway interpretation
• Visualization of NGS outputs using basic plots and genome browsers
• How to prepare an NGS analysis summary report
• Common mistakes in NGS data interpretation

Hands-On Activity:

• Review sample alignment statistics
• Interpret a sample VCF or RNA-Seq count result
• Prepare a simple NGS interpretation summary table
• Draft a mini NGS data analysis report

Hands-On Tools:

• SAMtools Awareness,IGV Genome Browser Awareness,Excel / Google Sheets,Sample VCF / Count Matrix,Google Colab

• Undergraduate and postgraduate students from Biotechnology, Bioinformatics, Genetics, Genomics, Molecular Biology, Microbiology, Biochemistry, Life Sciences, Pharmacy, Biomedical Sciences, and related fields
• PhD scholars and research scholars beginning work with sequencing data
• Faculty members and academicians interested in learning the basic NGS workflow
• Clinical research, biomedical, agricultural, microbiology, and biotechnology professionals
• Wet-lab researchers who want to start learning bioinformatics and sequencing data analysis
• Beginners in bioinformatics who want practical exposure to FASTQ files, quality control, read preprocessing, and biological interpretation