高通量测序技术在静脉吸毒人群HCV gt3感染者中的应用和研究

High-throughput next generation sequencing technology for the comprehensive assessment of full-length hepatitis C viral genomes in IDU population of Guangdong province

目的针对广东地区静脉吸毒人群中流行的HCV gt3建立高通量二代测序技术,以期获得HCV gt3全基因组序列用于流行病学研究。方法随机选取10例病毒滴度>10~6 IU/mL以及10例病毒滴度<10~6 IU/mL的标本进行高通量二代测序方法的探讨。通过样品均质化、过滤和核酸酶消化等前处理,对RNA提取方法进行比较,对不同滴度的标本采用不同的建库方法。采用"比对到HCV参考序列"和"宏基因组"2种生物信息学方法进行分析获得全基因组序列,将分型结果与前期NS5B和E1进行比较。结果磁珠法比柱提法获得的HCV RNA产出量高(79.428%vs 38.462%),富集法获得的"HCV reads/reads"的比例远远大于直接建库法(P<0.05),当病毒滴度>10^6 IU/mL时,用富集法和直接建库法获得的基因组覆盖度无差异(P>0.05)。当病毒滴度<10^6 IU/mL时,富集法获得的基因组的覆盖度远远大于直接建库法(P<0.05)。获得的全基因组序列的基因分型与NS5B和E1的基因分型一致。结论对于HCV gt3的NGS测序方法,RNA提取采用磁珠法,对于病毒滴度>10^6 IU/mL的标本,采用直接建库法可以获得全基因组序列,而对于病毒滴度<10^6 IU/mL的标本,只有采用富集法才能获得全基因组序列。

Objective To establish the high-throughput next generation sequencing for HCV gt3 in intravenous drug use(IDU) population of Guangdong province to obtain HCV full-length genomes.Methods 10 samples with the viral load greater than 10^6IU/mL and 10 samples with viral load lower than 10^6IU/mL were radomly selected. Virus particle processing steps included sample homogenization, filtration, ultra-centrifugation, nuclease digestion, RNA extraction method and enrichment by HCV specific probes. Two bioinformatic analysis methods, that is HCV reference sequences and metagenomics, were used to generate the HCV full-length genomes for phylogenetic tree construction. The genotype results from HCV full-length genomes and NS5 B region or E1 region were compared. Results For nucleic acid extractions, magnetic beads improved viral RNA output in comparison to silica spin columns(79.428% vs 38.462%). Enrichment method can get higher "HCV reads/total reads" than directed NGS library preparation method(P<0.05). When viral load were greater than 10^6IU/mL, there was no difference in HCV genome coverage between enrichment method and directed NGS library preparation method(P>0.05). In adverse, the genome coverage of enrichment method was significant higher than directed NGS library preparation method(P<0.05). The genotype of HCV full-length genome was in consist with NS5 B region and E1 region. Conclusion When the viral load was greater than 10^6IU/mL, directed NGS library preparation method can be used to get HCV full-length genome using magnetic beads as nucleic acid extraction. When the viral load was lower than 10~6IU/mL, enrichment method can be used to get HCV full-length genome.