On-site, high-fidelity target sequencing and absolute quantitation for HIV-1 surveillance

项目总结/摘要 联合国艾滋病规划署制定了雄心勃勃的90/90/90目标，以结束艾滋病毒/艾滋病的流行：到2020年，90%的 艾滋病毒感染者将知道自己的艾滋病毒状况; 90%的确诊者将接受持续的抗逆转录病毒治疗 治疗; 90%的治疗者将实现病毒抑制。实现这些目标将 需要对重点人群进行精确有效的检测、患者护理和预防干预， 在资源有限的环境中。微生物病原体的遗传多样性-不仅仅是HIV-1， 正在开发/使用抗微生物药物的病原体-仍然是那些 努力控制它们。耐药性监测和微生物病原体的基因分型， 长期以来一直受到有限读长和错误测序的困扰。拟议项目将开发一个 创新的测序和定量平台技术，可以灵敏地检测和准确地 通过条形码介导的全基因组测序，使用所谓的 “第三代”纳米孔测序技术（目的1和2）。利用口袋大小 MinION纳米孔测序仪和其他便携式生物技术设备的移动性， 我们将进一步开发这项技术，用于现场和实时监测， 即使在资源有限的环境中，也能对新出现的病毒进行耐药性诊断（目标3）。成功完成 这项研究将产生一种低成本，超高精度的测序和定量技术， 适用于科学和医学的不同领域的问题，从从头基因组组装到点， 护理耐药基因分型HIV-1为这项技术提供了一个很好的测试平台，因为它具有很高的 患者内遗传多样性以及现场和全基因组基因分型的需要。

PROJECT SUMMARY / ABSTRACT UNAIDS has set ambitious 90/90/90 targets toward ending the HIV/AIDS epidemic: that by 2020, 90% of people living with HIV will know their HIV status; 90% of those diagnosed will be on sustained antiretroviral treatment; and 90% of those on treatment will have achieved viral suppression. Reaching these goals will require precise and efficient testing, patient care, and prevention interventions for key populations, particularly in resource-limited settings. The genetic diversity of microbial pathogens – not just HIV-1 but any number of pathogens for which antimicrobial drugs are being developed/employed – remains a major challenge for those endeavoring to control them. Drug-resistance surveillance and the genotyping of microbial pathogens have long been plagued by limited read lengths and erroneous sequencing. The proposed project will develop an innovative sequencing and quantitation platform technology that can sensitively detect and accurately delineate HIV-1 variants in patients through barcode-mediated, whole-genome sequencing using the so-called “third-generation” Nanopore sequencing technology (Aims 1 and 2). Taking advantage of the pocket-size mobility of the MinION Nanopore sequencer and other portable biotechnology devices that have recently became available, we will further develop this technology for on-site and real-time surveillance and drug- resistance diagnosis of emerging viruses even in resource-limited settings (Aim 3). Successful completion of this study will produce a low-cost, ultra-high-accuracy sequencing and quantitation technology with broad applicability to problems in diverse areas of science and medicine, from de novo genome assembly to point-of- care drug-resistance genotyping. HIV-1 offers an excellent testbed for this technology due to the high degree intra-patient genetic diversity and the need for on-site and genome-wide genotyping.