NadPrep RNA & DNA Library Co-Preparation Module Infectious diseases caused by pathogenic microorganisms are among the significant causes leading high mortality and morbi- dity in humans. The types of pathogenic infections are diverse, including parasites, fungi, bacteria, mycoplasma/chlamydia/spirochetes/Rickettsia, viruses, etc. Specific diagnostic and therapeutic measures are often required for different pathogenic infections. Therefore, rapidly and accurately detecting the causative pathogen is crucial for the diagnosis, treatment, and manage- ment of infectious diseases. DNA detection process is suitable for suspected DNA pathogen infections, while RNA detection process is applicable for suspected RNA virus infections. When viral infection are challenging to exclude, it is recommended to simultaneously perform RNA and DNA co-detection. However, the dual-process detection of RNA and DNA is costly , and there is an urgent need for an RNA & DNA single-tube library co-preparation scheme to reduce detection costs and shorten detection time. Combining targeted next-generation sequencing (tNGS) of pathogenic microorganisms further aids in rapid and accurate clinical diagnosis and treatment of infections. To expedite the entire process of pathogenic microorganisms detection in tNGS, Nanodigmbio has launched NadPrep RNA & DNA Library Co-Preparation Module, enabling a single process for RNA & DNA codetection. NadPrep RNA & DNA Library Co-Preparation Module is developed for high-throughput sequencing platforms, enabling a single process for RNA & DNA co-preparation from mixed pathogenic microbial samples. This module consist of 1st Strand Synthesis, 2nd Strand Synthesis, Fragmentation and End Repair & A-Tailing, Adapter Ligation and Amplification. It can be used in conjunction with the NadPrep Universal Stubby Adapter (UDI) Module and NadPrep Universal Adapter (MDI) Module (for MGI) for different sequencing platforms. Flexible Compatibility Supports mixed samples with varying ratios of 10-100 ng RNA and 1-100 ng DNA Compatible with both MGI and Illumina sequencing platforms Single-tube operation, quick and convenient RNA & DNA library co-preparation in a single process in 3-3.5 hours High efficiency and stability of library yield Uniform size distribution of fragment Background Pathogenic Microorganism Control ●Comprehensive control of background contamination of pathogenic microorganisms throughout the entire process, including environmental control, consumables control, raw material control, process control, manufacturing process control, quality

Mixed Sample RNA Degeneration & Random Primer Hybridization Fragmentation / End Repair & A-tailing Adapter Ligation Post-ligation Cleanup Co-Random Primer Index Index Index Index Co-Random Primer Index Index Post-amplification Cleanup NadPrep Universal Stubby Adapter Index Index Index Index

Flexible Compatibility Figure 1. NadPrep RNA & DNA Library Co-Preparation Module for library co-preparation with mixed RNA & DNA samples in varying initial ratios. A. Library yield (MDI); B. Library yield (UDI). Utilize NadPrep RNA & DNA Library Co-Preparation Module in conjunction with NadPrep Universal Adapter (MDI) Module (for MGI) and NadPrep Universal Stubby Adapter (UDI) Module for library co-preparation. Note: The RNA samples are from K562 cell lines; the DNA samples are Human Genomic DNA Standards (Promega, G1521). Reads distribution of rRNA (lg) Figure 2. Detection of tNGS Total Solution...

tNGS mNGS Figure 3. Distribution of detected reads in simulated pathogenic microbial samples using tNGS and mNGS based on the NadPrep RNA & DNA Library CoPreparation Module. A. Reads distribution of Coronavirus; B. Reads distribution of Candida albicans. Note: Sequencing platform: Illumina NovaSeq 6000, PE150. Figure 4. Capture performance of mixed RNA samples in varying copy numbers with co-preparation and RNA-preparation. A. Reads distribution; B. Enrichment fold. Note: The X-axis represents the copy number of the Reference material for SARS-CoV-2 Omicron Variant Genomic RNA. The samples are...

Normalized coverage Staphylococcus aureus Normalized coverage Normalized coverage Figure 6. Uniform coverage of regions with various GC content using NadPrep RNA & DNA Library Co-Preparation Module. A. Staphylococcus aureus, B. Bacillus subtilis, and C. Lactobacillus fermentum with different GC content. Application Example in Clinical Sample A Figure 7. Detection performance of tNGS Total Solution for clinical samples with NadPrep RNA & DNA Library Co-Preparation Module. A. Library yield; B. Reads ratio of Rhinovirus A and Rhinovirus C. Note: The samples were derived from 50 ng of clinical rhinovirus...