2) Next Generation Sequencing (NGS) - Sample Preparation

Introduction to Next Generation Sequencing concepts and the four main NGS application types: WGS (whole genome sequencing), Exome-Seq, RNA-Seq, and Methyl-Seq. Overview of why sample preparation is critical before sequencing and library preparation from genomic DNA or total RNA.

Detailed protocols for four WGS library kits: TruSeq PCR-free (1-2 μg DNA), TruSeq Nano (100-200 ng DNA), Nextera (large genomes), and Nextera XT (small genomes/bacteria). Includes DNA fragmentation, end repair, adenylation, adapter ligation, and validation by qPCR or Bioanalyzer.

Two exome-seq approaches: Nextera Rapid Capture Exome Kit for exons only, and Extended Exome Kit for complete analysis including UTRs and miRNA regions. Process involves tagmentation, PCR enrichment, and biotin-streptavidin-based exome capture with magnetic bead purification.

TruSeq Stranded Total RNA protocol: deplete ribosomal RNA using magnetic beads, fragment remaining RNA, synthesize first-strand cDNA with actinomycin D, synthesize second-strand cDNA using dUTP nucleotides, ligate adapters, and perform PCR enrichment with validation by qPCR.

TruSeq Stranded mRNA Kit: mRNA enrichment instead of rRNA depletion. TruSeq Small RNA Kit: blunt-end adapter ligation (3' then 5' adapters), RT-PCR enrichment, agarose gel electrophoresis for 147-157 bp selection, and Bioanalyzer validation.

TruSeq DNA Methylation Kit: fragment genomic DNA, convert unmethylated cytosines to uracil using bisulfite while preserving methylated cytosines, amplify DNA with random primers containing 5' adapter sequences, ligate 3' adapter tags, perform PCR enrichment, and validate by qPCR or Bioanalyzer.