Do high fidelity enzymes need to be used in conjunction with dNTPs for NGS library amplification?

The uniformity and yield of NGS library construction, especially 16S amplicons, have a significant impact on the quality of subsequent sequencing data. The high fidelity enzyme manual recommends matching dNTPs, usually the manufacturer has optimized the concentration, purity, and buffer ions of dNTPs to reduce amplification preferences and mismatches. Non matching dNTPs, if purity pH、 Concentration deviation may indeed lead to changes in the amplification efficiency of certain templates, ultimately resulting in a decrease in library uniformity. The supplier said that ordinary dNTPs can be used and the basic ingredients are correct, but 'usable' and 'suitable for NGS' are two different things. If you want to create a multi sample 96 well library, I do not recommend saving this money on key reagents. You can first ask the technical support of the enzyme manufacturer to confirm whether they accept dNTPs from other brands, in order to avoid after-sales disputes in case of problems.

If the budget is tight, consider two approaches: first, ask the enzyme manufacturer whether it has domestic or economy-tier dNTPs validated for use with its kit; second, run validation with domestic dNTPs, but do not substitute them directly. When selecting domestic alternatives, focus on HPLC purity, nuclease-free status, endotoxin-free status, batch-to-batch consistency, and supply lead time. You can also run a preliminary round using samples provided by the supplier. Acceptance criteria include library yield no less than 80%-90% of that of the kit-matched group, consistent main fragment peak, and clean NTC. After passing acceptance, proceed with bulk purchasing and keep the validation data on file. For 16S amplicon workflows, the pre-processing steps and PCR cycle number also affect uniformity, so do not focus only on dNTPs.

Imported brand high fidelity dNTPs with a capacity of 250 μ L per tube, with prices ranging from several hundred to thousands of RMB per tube depending on the brand and concentration. Please consult the agent for specific details. High fidelity enzyme matching dNTPs are often more expensive than ordinary dNTPs because of their higher purity and quality control indicators. In terms of delivery time, if the 250 μ L package is in stock, it can generally arrive within one week, while non stock or large packaging may take 2-6 weeks. Build a library with 96 samples at a time. The amount of dNTPs used is usually not large, but the PCR cycles and samples in the amplicon library are large. It is recommended to leave an additional 20% -30% margin for purchase based on the actual number of reactions. Simultaneously inquire about the batch and expiration date, and it is best to complete the NGS project with the same batch of reagents.

To verify whether non-matched dNTPs cause a decrease in library yield or uniformity, it is best to perform a small-scale parallel experiment: use the same batch of 16S samples, the same primers, and high-fidelity enzyme, and construct libraries with matched dNTPs and regular dNTPs separately. Compare library concentration and fragment distribution (Bioanalyzer or electrophoresis), and if possible, run a small chip to examine alpha diversity and the relative abundance of major microbial taxa. If the non-matched group shows lower yield, shifted fragment size, or loss of low-abundance templates, this indicates a risk. I encountered a case where switching to regular dNTPs reduced library yield by about 20-30%, which was not a complete failure, but the uniformity of the sequencing data worsened. It is recommended to test at least 8-16 samples before running the full 96 samples.