Best Practices for Nextera Library Prep Expert Video Tip

Evaluate the quality and quantity of input DNA using fluorometric methods like Qubit or Picogreen, avoiding UV absorbance. Check purity ratios (260/280 >1.8 and 280/230 >2.0) and perform additional purification if needed to remove contaminants.

Incubate DNA with the modified transposon to simultaneously cleave and tag the DNA with partial adapter sequences. Ensure accurate DNA input to avoid under-tagmentation (too much DNA) or over-tagmentation (too little DNA).

Learn how excess DNA input or enzymatic inhibitors (EDTA, proteins, detergents, phenols) lead to under-tagmentation and inefficient clustering. Use proper quantification and purification methods to prevent larger-than-optimal library fragments.

Identify three main causes of over-tagmentation: insufficient DNA input, degraded DNA (FFPE samples), and small amplicons. For amplicon libraries, use DNA fragments at least 300 base pairs with primers designed 50+ base pairs upstream and downstream.

Run tagmented samples on a high sensitivity bioanalyzer to verify library size distribution (200 base pair to 1.5 KB). Optionally run samples after PCR amplification before bead cleanup to confirm fragments exceed 200 base pairs.

Use fluorometric methods (Qubit or Picogreen) to quantify double-stranded DNA after PCR cleanup. Do not use bioanalyzer traces or qPCR for quantification due to broad library distribution.

For Nextera XT kits, use bead-based normalization by adding libraries to normalization beads; elute normalized single-stranded libraries that can be pooled at 1:1 volume ratio. Store double-stranded libraries frozen before normalization, and keep single-stranded libraries at -20°C for up to one week.

Consult Illumina's technical support pages, troubleshooting technical notes, and field application scientists for additional guidance on Nextera library preparations.