Optimized MT-Capture targeted enrichment sequencing for the complete genome of norovirus in stool and sewage samples.

Human norovirus is a major acute gastroenteritis pathogen with high genotypic diversity, mutation, and recombination rates. Existing sequencing methods for monitoring its genetic variations are limited by low sensitivity and contamination risk. This study optimized our previously established Micro Target Hybrid Capture System (MT-Capture) by increasing nucleic acid input, streamlining library preparation, and introducing a deployable USER enzyme-based contamination prevention system. The optimized protocol completes library preparation and capture within 7 hours. Compared with multiplex PCR sequencing, optimized MT-Capture avoids primer updates, captures multiple genotypes with improved compatibility, and detects low-abundance samples more efficiently. The USER enzyme degrades contaminating amplicons, ensuring data authenticity. The MT-Capture-USER system provides a rapid, sensitive, and contamination-resistant approach for norovirus whole-genome sequencing and epidemiological surveillance.