Next Generation Sequencing 2: Illumina NGS Sample Preparation - Eric Chow (UCSF)

Overview of how Illumina sequencing works in flow cells using optical detection of color-labeled bases across multiple cycles. Explains how adapters are added to DNA inserts to enable capture and sequencing of library molecules.

Fragment long DNA into appropriately-sized pieces, blunt the ends, add single A overhangs, and ligate colored adapters with T overhangs. Optionally amplify via PCR or proceed as PCR-free library for sequencing.

Use transposase enzymes loaded with adapter sequences to simultaneously fragment genomic DNA and insert partial adapters in a single reaction. Complete adapter sequences via PCR amplification.

Select regions of interest using capture-based methods with biotin-labeled DNA probes pulled down by magnetic beads, or amplify targets using multiplexed PCR primers with partial Illumina adapters followed by complete adapter ligation.

Select messenger RNA transcripts using either poly-A tail capture with oligo-dT coated beads, or deplete ribosomal RNA using biotin-labeled probe panels pulled down by magnetic beads. Elute enriched mRNA for library preparation.

Fragment enriched mRNA into appropriately-sized small fragments suitable for Illumina sequencing. Convert RNA fragments to complementary DNA for compatibility with Illumina sequencers.