What should be done if the starting DNA amount is insufficient for NGS library preparation in tumor mutation detection?

Although a starting amount of 10-30 ng is lower than the 50 ng specified in the instructions, many low-input NGS library preparation kits can actually handle 10 ng or even less. It is recommended to first choose products labeled for 1-50 ng input or single-cell-level library preparation, rather than simply increasing the number of PCR cycles yourself, which can easily lead to elevated duplication rates and false positives. First, use 1-2 samples with known mutations to perform a gradient starting-amount pilot experiment, such as preparing libraries at 50, 30, and 10 ng separately, and check whether library yield, complexity, coverage uniformity, and mutation detection are consistent. If the complexity of the 10 ng library decreases significantly, continue optimizing extraction or pooling similar samples rather than blindly proceeding to clinical use.

If you're in a hurry to get started, first contact the reagent vendor to ask about stock availability. For imported single-cell-level or low-input library preparation kits, lead times are generally 2–6 weeks; some domestic low-input kits can ship in 1–2 weeks, depending on inventory and cold-chain logistics. You can ask the supplier to provide a loaner pack or trial pack, and first run through the workflow using standards. Before starting, be sure to include negative controls and known positive standards to monitor background noise and false-positive rates; if the input amount is only 10 ng, it is recommended to include one low-input positive quality control and one no-template control with every batch to confirm that neither contamination nor amplification bias affects interpretation. After the kit arrives, don't rush into clinical use—complete at least one batch of methodological validation first.

<p>For micro-biopsy samples, the extraction yield is low, making the extraction step more worthy of optimization than library preparation. Pancreatic cancer tissue typically shows significant nucleic acid degradation. It is recommended to use a magnetic bead-based method instead of precipitation or column-based methods to reduce tube transfer and drying losses, and to minimize the elution volume to 20-40 μL. Before library preparation, take 1-2 μL for Qubit quantification and fragment size distribution analysis to confirm the proportion of small fragments; if the fragments</p>