Sequencing & Library Preparation

May 27, 2025 · Mia Wu

Overview

Next-generation sequencing (NGS) , a transformative technology in clinical and biomedical research, offers high sensitivity and specificity for both qualitative and quantitative analyses, widely applied in pathogen detection, cancer genomics, and reproductive genetics.

NGS workflow

Next-generation sequencing involves four steps:

Step1 Nucleic Acids Extraction :

During extraction, nucleic acids (DNA or RNA) are isolated from a sample by lysing cells and purifying the genetic material from other cellular components. 

Step2 Library preparation :

DNA or RNA is fragmented, and sequencing adapters are ligated to the fragments to facilitate amplification and compatibility with the sequencing platform.

Fragmentation and end repair: DNA is fragmented and end-repaired to ensure compatibility with sequencing adapters.

Addition of adapters: Adapters, which may include barcodes for sample identification and multiplexing, are attached after or during fragmentation.

PCR amplification (optional): PCR is used to amplify library DNA when input is low or required by the protocol. It enriches fragments with proper adapters but may introduce duplication or bias, especially in GC-rich regions. PCR-free methods are preferred for high-fidelity applications if input DNA is sufficient. Post-PCR cleanup removes residual primers and adapter dimers.

Step3 sequencing:

NGS platforms sequence millions of DNA fragments in parallel, generating large volumes of data. The read length and depth vary depending on the sequencing platform and reagent kit used.

Step4 Alignment and Data analysis:

Reads are filtered, aligned to a reference genome, and analyzed to identify and annotate variants using specialized software.