Objective To investigate the application of next-generation sequencing(NGS) in determining the full-length sequence and baseline resistance-associated substitution(RAS) of hepatitis C virus(HCV) subtype 3 b. Methods Nucleic acid was extracted from plasma of a HCV RNA-positive blood donor,and after sequence-independent amplification,a sequencing library was constructed and NGS was performed using Illumina Hiseq. Bioinformatics methods were used to analyze full-length HCV sequence,viral genotype,and baseline RAS to direct-acting antivirals(DAAs). Results A total of 8. 4 Gb data with more than 56 million reads were obtained. The full-length HCV sequence was obtained by bioinformatics analysis,with an average sequencing depth of 488 007 and a genotype of 3 b subtype. A total of 12 RASs were identified in HCV amino acid sequence,i. e.,Y56 H,Q80 K,Q80 R,and A156 G located in NS3,M28 G,Q/A30 G,Q/A30 K,L31 F,L31 M,and Y93 H located in NS5 A,and S282 T and V321 A located in NS5 B,among which Q/A30 K and L31 M located in NS5 A had high frequencies of 99. 16% and 98. 37%,respectively,while the other 10 RASs had low frequencies of < 0. 5%.Conclusion NGS can be used to determine the full-length sequence and genotype of HCV subtype 3 b and identify baseline RASs,which has great significance in the epidemiological study of HCV subtype 3 b and the development of DAA treatment regimens. [ABSTRACT FROM AUTHOR]