HCV Genotyping with 5'NCR and NS5B regions with next generation sequencing (YND-NGS)

ABSTRACT Introduction and AIM: Hepatitis C virus (HCV) has very high genetic diversity. HCV strains are divided into seven known genotypes based on phylogenetic and sequence analysis of viral genomes. Genotype identification of HCV is important in determining the appropriate treatment of the patient and the duration of the treatment. In particular, direct identification of genotypes and subtypes have become even more important as direct-acting antivirals become available for treatment. Nucleic acid sequence analysis of the whole genome is the gold standard for HCV genotyping. In practice, NS5B, core, and 5'NCR regions are analysed for genotyping. HCV genotype test has been routinely performed since 2000 in the Molecular Virology Laboratory of the Department of Medical Microbiology, Faculty of Medicine, Ege University. HCV genotyping with Sanger chain termination reaction is currently routinely being performed from HCV positive patient samples. In this study it is aimed to evaluate the eficiency of genotyping by using HCV 5'NCR and NS5B regions with next generation sequencing (NGS) and to determine the appropriate HCV genotyping method in our laboratory. Materials and methods: Sixty archived (at -80°C) HCV positive plasma samples which were sent for HCV genotyping to the laboratory were included in the study. RNA extraction, reverse transcription steps followed by external PCR, nested (second round) PCR and analysis steps for the 5'NCR region and NS5B region separately. PCR products of the 5'NCR and NS5B regions of the HCV genome were analyzed with the ABI 3500 Genetic Analyzer (Applied Biosystems Foster City, CA, USA) and the MiSeq (Illumina, San Diego, CA, USA) (NGS), and the nucleic acid sequences were genotyped at the NCBI gene bank (https://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) genotyping tool. Results: According to the analysis of data obtained with NGS; by NS5B region sequence analysis 43 (72%) of the 60 samples were genotype 1b, seven (11%) genotype 4, four (7%) genotype 3, three (5%) genotype 1a, two (3%) genotype 5, one (2%) was genotype 2. With the 5'NCR region sequence analysis; 36 (60%) of the 60 samples were genotype 1b, seven (12%) genotype 4, four (7%) genotype 3, five (8%) genotype 1a, two (3%) genotype 5, two (3%) genotype 6, one (2%) was genotype 2. With 5'NCR region sequence analysis, three samples (5%) were identified as genotype 1 without subtype discrimination. According to analysis of both regions, genotype 1b was the most detected genotype. There was no sample defined as genotype 7. With NGS, 38-40 base longer clean sequences could be obtained compared to the Sanger sequences. When Sanger and NGS sequences were aligned on sample basis; among the 5'NCR region sequences, 53 (88,3%) of the 60 samples had 100% identity, five of them (8,3%) had 99% identity, one of them (1,6%) had 98% identity, one of them (1.6%) had 97% identity. Among the NS5B region sequences, 45 (73.3%) of the 60 samples had 100% identity, five (8.3%) had 99% identity, one (1.6%) had 98% identity, four (6.6%) had 95-97% identity, 4 (6.6%) had 92-94% identity, one (1.6%) had 83% identity. The 83% identical sample was determined as genotype 1a with Sanger and as genotype 1b with NGS. This sample was identified as genotype 1a by 5 'NCR sequence analysis both with NGS and Sanger. Sanger chain termination reaction and NGS data in both groups showed that; three samples identified as genotype 1, while subtype discrimination could not be done with 5'NCR region sequence analysis, these samples were identified as genotype 1b with NS5B region sequence analysis. Two samples were identified as genotype 1a with 5'NCR region sequence analysis, but identified as genotype 1b with NS5B region sequence analysis. Two samples were identified as genotype 6 with 5'NCR region sequence analysis but identified as genotype 1b with NS5B region sequence analysis. It was thought to be associated to the insufficiency of 5'NCR seuence for subtype determination and discrimination of genotype 6 from some genotype 1 strains as it has been known. One sample was identified as genotype 1b by NS5B region sequence analysis with the Sanger chain termination reaction, but identified as genotype 1a by 5'NCR region sequence analysis with Sanger, NS5B region sequence analysis and 5'NCR region sequence analysis with NGS. The NS5B sequence which could be obtained by Sanger method was short; it was thought to be the reason of incompatibility. Conclusion: Next generation sequencing and Sanger chain termination results were the same both with NS5B and 5'NCR regions in 59 of 60 samples (98.33). Cost was reduced with NGS by pooling and "multiplexing" the samples. Analysing NS5B region with a reliable method such as NGS is efficient in determining the genotypes which is clinically important to guide the treatment. According to overall evaluation of the data; 43 of 60 samples (71.67%) were genotyped as genotype 1b, three were genotype 1a (5%), seven were genotype 4 (11.67%), four were genotype 3 (6.67%), two were genotype 5 (3.33%), an done was genotype 2 (1.67%). Key words: HCV genotyping, Next Generation Sequencing, NS5B, 5'NCR