NGS Library Preparation
Related: DNA Extraction · Whole-Genome Sequencing · Targeted Enrichment · Sequencing Technologies · Nucleic Acid Quantification
1. Principle
Library preparation converts extracted nucleic acid into sequencer-ready fragments with platform-specific adapters, barcodes, and (optionally) unique molecular identifiers (UMIs), so millions of molecules can be read in parallel.
2. Step-by-Step Procedure (conceptual)
- Quantify/qualify input DNA/RNA (Nucleic Acid Quantification).
- Fragment (enzymatic, mechanical, or tagmentation) to desired insert size.
- End-repair / A-tailing (Illumina-style) as required.
- Ligate adapters ± sample barcodes / UMIs.
- Size-select and amplify (PCR cycles minimized to reduce bias).
- Final QC (molarity, size) → pool → sequence (Whole-Genome Sequencing / Metagenomic NGS).
- For RNA: reverse transcription + second strand before or via RNA-seq kits; for ONT: rapid/ligation kits differ.
3. Interpretation / QC
- Wrong insert size → poor clustering/yield.
- Over-amplification → PCR duplicates (UMIs help).
- Index hopping / mis-assignment — use dual indexes when possible.
- Contaminated inputs → garbage genomes (Contaminant and Mixed-Culture Detection).
4. Clinical Use Cases
- Bacterial isolate WGS for outbreaks and resistome.
- Viral WGS (HIV, SARS-CoV-2) after amplicon or hybrid enrichment.
- mNGS libraries from sterile-site specimens.
5. Comparison with Other Methods
| Path | Use |
|---|---|
| Shotgun WGS library | Pure isolates |
| Amplicon library | Tiled viral genomes / 16S |
| Targeted Enrichment | Low-load pathogens in host DNA |
6. Mnemonic / Visual Aid
Cut → Tag → Amplify → Sequence
Related Concepts
Active Recall
- What problem do UMIs address?
- Why minimize PCR cycles in library prep?
- How does tagmentation combine fragmentation and adapter addition?