背景:在临床管理中建议进行HIV-1耐药性测试,并且现在许多病毒学实验室都可以使用下一代测序(NGS)方法。
目的:评估长阅读单分子实时(SMRT)测序的诊断性能(Sequel,PacBio)用于HIV-1聚合酶基因分型。
方法:使用Sanger测序对111个前瞻性临床样本(83个血浆和28个富含白细胞的血液部分)进行了常规HIV-1抗性基因分型分析,VelaNGS,和SMRT测序。我们开发了SMRT测序方案和生物信息学管道来推断单倍型和变体调用方法的抗逆转录病毒抗性。
结果:在98%的血浆RNA样品中,病毒载量高于4log拷贝/mL时,通过三个平台成功测序了聚合酶。对于3至4个对数拷贝/mL的病毒载量,使用Sanger或Vela测序的成功率降至83%,使用SMRT测序的成功率降至67%。灵敏度为50%,54%和61%是使用SMRT获得的,Vela,和Sanger测序,分别,在血浆中检测不到HIV-1RNA的患者的细胞DNA中。使用SMRT测序检测到通过Sanger测序鉴定的98%的抗性相关突变(RAM)。此外,使用SMRT测序检测到用VelaNGS鉴定的91%的RAM(>5%阈值)。使用Vela和SMRT测序的RAM定量具有良好的相关性(Spearman相关性ρ=0.82;P<0.0001)。
结论:全长HIV-1聚合酶的SMRT测序在表征RNA和DNA临床样本上的HIV-1基因型抗性方面表现良好。长读测序是突变单倍型和抗性分析的新工具。
BACKGROUND: HIV-1 resistance testing is recommended in clinical management and next-generation sequencing (NGS) methods are now available in many virology laboratories.
OBJECTIVE: To evaluate the diagnostic performance of Long-Read Single Molecule Real-time (SMRT) sequencing (Sequel, PacBio) for HIV-1 polymerase genotyping.
METHODS: 111 prospective clinical samples (83 plasma and 28 leukocyte-enriched blood fraction) were analyzed for routine HIV-1 resistance genotyping using Sanger sequencing, Vela NGS, and SMRT sequencing. We developed a SMRT sequencing protocol and a bio-informatics pipeline to infer antiretroviral resistance on both haplotype and variant calling approaches.
RESULTS: The polymerase was successfully sequenced by the three platforms in 98 % of plasma RNA samples for viral loads above 4 log copies/mL. The success rate decreased to 83 % using Sanger or Vela sequencing and to 67 % using SMRT sequencing for viral loads of 3 to 4 log copies/mL. Sensitivities of 50 %, 54 % and 61 % were obtained using SMRT, Vela, and Sanger sequencing, respectively, in cellular DNA from patients with prolonged undetectable plasma HIV-1 RNA. Ninety-eight percent of resistance-associated mutations (RAMs) identified with Sanger sequencing were detected using SMRT sequencing. Furthermore, 91 % of RAMs (> 5 % threshold) identified with Vela NGS were detected using SMRT sequencing. RAM quantification using Vela and SMRT sequencing was well correlated (Spearman correlation ρ = 0.82; P < 0.0001).
CONCLUSIONS: SMRT sequencing of the full-length HIV-1 polymerase appeared performant for characterizing HIV-1 genotypic resistance on both RNA and DNA clinical samples. Long-read sequencing is a new tool for mutation haplotyping and resistance analysis.