NadPrep EZ RNA & DNA Library Co-Preparation Module: Fast, More Than Just a Workflow; Accurate, More Than Just Detection

Have you ever encountered these frustrating issues during the preparation of libraries for pathogen detection？

Don’t panic~

The NadPrep EZ RNA & DNA Library Co-Preparation Module has got you covered!

01 Introduction

The NadPrep EZ RNA & DNA Library Co-Preparation Module features a premixed component design that significantly reduces reagent handling and pipetting steps. It also eliminates 2^nd-strand cDNA synthesis and post-ligation cleanup, further accelerating the library preparation process. The module enables simultaneous metagenomic library preparation of mixed RNA and DNA samples within a single reaction system while remaining fully compatible with liquid-phase hybridization target capture sequencing.

Figure 1. Workflow and turnaround-time comparison of the two co-library preparation Kits.

Note： Co-Prep refers to the NadPrep RNA & DNA Co-Library Preparation Kit, while EZ Co-Prep refers to the NadPrep EZ RNA & DNA Library Co-Preparation Kit.

02 Feature

2.1 Flexible

• Supports simultaneous lib prep from mixed nucleic acid samples containing 1-400 ng RNA and/or DNA across a wide range of input ratios.

• Fully compatible with mainstream sequencing platforms such as NovaSeq and DNBSEQ, with excellent automation adaptability.

2.2 Stable

• Optimized enzymatic system and reaction conditions ensure robust and reproducible library generation.

• Uniform library size distribution and low GC bias minimize coverage bias, ensuring stable and reliable performance.

2.3 Fast

• Ready-to-use premixed components combined with a single-tube workflow minimizes reaction setup and pipetting steps.

• Optimized workflow eliminates 2^nd-strand cDNA synthesis and post-ligation cleanup steps, reducing the total time within 3 hr.

2.4 Reliable

• ISO 13485-certified manufacturing with comprehensive background microbial control minimizes microbial residues and reduces the risk of false-positive results.

• Establishes and continuously updates an in-house background microbial database covering the entire workflow to effectively identify and exclude exogenous microbial interference.

03 Performance

3.1 Flexible Compatibility · Consistent Efficiency

The NadPrep EZ RNA & DNA Library Co-Preparation Module is compatible with both NovaSeq and DNBSEQ sequencing platforms. Across a total input range of 1-400 ng, it consistently delivers stable library yields, meeting the requirements of downstream applications such as metagenomic sequencing and hybrid-capture sequencing. Meanwhile, the resulting library fragments are predominantly distributed within 250–500 bp, consistent with the expected fragment-size range.

Figure 2. Library preparation performance of the NadPrep EZ RNA & DNA Library Co-Preparation Kit for mixed RNA and DNA samples across different input amounts. A. Library yield; B. Size distribution of the libraries (NovaSeq).

Note：RNA samples were extracted from the Jurkat cell line. DNA samples were Human Genomic DNA standards (Promega, G1521).

3.2 Strict Background Control · Reliable Results

The NadPrep EZ RNA & DNA Library Co-Preparation Kit maintains extremely low levels of residual background microorganisms, effectively minimizing reagent-derived background interference and providing a reliable data foundation for the accurate identification and interpretation of suspected pathogenic microorganisms in clinical samples.

Table 1. Evaluation of microbial residues in the NadPrep EZ RNA & DNA Library Co-Preparation Kit using an in-house background microbial database.

Note：The host-mimicking mixed nucleic acid samples consist of Human Brain Total RNA (Clontech, 636530) and Human Genomic DNA standard (Promega, G1521) mixed at 50 ng:50 ng. Sequencing was performed on NovaSeq 6000 with PE150. The average raw data was 5 M reads pair per sample.

3.3 Ultra-high Sensitivity · Accurate Detection

Compared with the NadPrep RNA & DNA Co-Library Preparation Kit (Co-Prep), the NadPrep EZ RNA & DNA Co-Library Preparation Kit (EZ Co-Prep) further broadens the template input range and enhances the detection capability for low-abundance pathogens.

Table 2. Comparison of pathogen detection performance between two RNA & DNA library co-preparation kits for reference standards across different concentration gradients.

Note：◉ Detected，◉Not detected. The maximum supported template volume specified in the respective user manuals was used as the input volume (12 μL vs. 22 μL). Hybrid capture was performed using the μCaler HyperMix Hybrid Capture Reagent and μCaler RIA Panel v1.0, with 0.5 Gb of raw data per sample was used for analysis.

3.4 Application Example in Clinical Sample

Using blood samples from individuals living with HIV, the NadPrep EZ RNA & DNA Co-Library Preparation Kit combined with the μCaler HIV-1 Panel generated hybrid-capture libraries that enabled high-coverage and highly consistent reconstruction of the full-length HIV-1 genome. Analysis of the pol gene further enabled the successful identification of multiple HIV-1 subtypes and circulating recombinant forms (CRFs), demonstrating the potential of this workflow for whole-genome analysis of individual pathogens.

Table 3. Library preparation performance and HIV-1 whole-genome sequencing capability of the NadPrep EZ RNA & DNA Library Co-Preparation Kit using blood samples from HIV-infected individuals.

04 Summary and Outlook

The newly upgraded NadPrep EZ RNA & DNA Co-Library Preparation Kit, centered on the concept of “one sample, single-tube co-detection, rapid library preparation,” enables simultaneous metagenomic library preparation from mixed RNA and DNA samples within 3 hr. This workflow substantially improves pathogen detection efficiency and the breadth of information obtained, facilitating comprehensive characterization of key pathogen features and providing enhanced technical support for the precision diagnosis and treatment of infectious diseases.