Establishment and preliminary application of a nanopore sequencing and tracking technology system based on a DENV-1 global unified genotyping collaborative monitoring strategy
Objective Constructed a nanopore sequencing traceability technology system based on the globally unified DENV-1 genotyping collaborative monitoring strategy and applied it preliminarily to the serum sample testing of patients with dengue fever. Methods Utilizing the DENV-1 whole genome sequences set in the Global Integrated Sequence and Genotyping Database for DENV (GISDD v1.2.1),the specific targeted multiplex PCR primer pairs were screened,designed,and evaluated using MEGA 7.0,Prime-Blast,MPprimer,and GISDDrPrimer software. The RNA from the serum samples was extracted and subjected to reverse transcription. The 16 pairs of primers were designed into 2 primer pools for multiplex PCR amplification of the target genome sequences. The library was constructed using magnetic bead purification and the SQK-LSK109 kit according to the operational procedure,followed by MinION sequencing. The sequencing data were analyzed and DENV-1 genomes were assembled for traceability analysis using the GISDD genetic typing platform. Results Sixteen pairs of DENV-1 primers with high coverage to the DENV-1 genomes were designed and identified,establishing a DENV-1 specific multiplex PCR-based nanopore high-throughput sequencing system. It was preliminarily applied to the whole-genome detection of serum samples from 13 patients with dengue fever in Guangzhou. The median of average reads length was 1668.00 bp,the median of average reads quality was 9.30,the median alignment rate for the reference genome was 97.90％,and the median length aligned to the reference genome NV46 was 1677.90 bp,with a median genome depth of 12886.40× . The subsequent genotyping and tracing analyses using GISDD revealed that 9 genomes belonged to DENV-1 clade 1E1,3 to 1L1,and 1 to 5C1. Conclusions The nanopore sequencing-based DENV-1 whole-genome detection system contextualizing within GISDD described here is characterized by its stability and reliability,enabling the acquisition of high-quality DENV-1 genomes. This approach offers an efficient technical solution for the rapid identification and traceability of DENV genomes during dengue outbreaks,thereby establishing a solid foundation for developing a global collaborative surveillance and control system for dengue.
万方数据
维普
