Identification of system-level features in HIV migration within a host

Objective: Identify system-level features in HIV migration within a host across body tissues. Evaluate heterogeneity in the presence and magnitude of these features across hosts. 
Method: Using HIV DNA deep sequencing data generated across multiple tissues from 8 people with HIV, we represent the complex dependencies of HIV migration among tissues as a network and model these networks using the family of exponential random graph models (ERGMs). ERGMs allow for the statistical assessment of whether network features occur more (or less) frequently in viral migration than might be expected by chance. The analysis investigates ve potential features of the viral migration network: (1) bi-directional ow between tissues; (2) preferential migration among tissues in the same biological system; (3) heterogeneity in the level of viral migration related to HIV reservoir size; (4) hierarchical structure of migration; and (5) cyclical migration among several tissues. We calculate the Cohran's Q stati..., Blood and tissue samples were collected during a rapid autopsy procedure.
Genomic DNA was extracted from 5 million PBMCs and snap-frozen tissues using QIAamp DNA Mini Kit (Qiagen cat#51306) per manufacturer's protocol. After extraction, precipitation was performed to concentrate DNA.
RNA was extracted from blood plasma layering 500-700µl of plasma on top of 200µl of 20% sterile filtered sucrose solution. Sample was spun at 23,500xg for 1 hour at 4°C to pellet the virus. Supernatant was removed and the pellet resuspended in 140µl of PBS. RNA was extracted using Qiagen’s QIAamp Viral RNA mini kit (cat# 52904) according to the manufacturer’s recommendation. cDNA from HIV RNA was generated using Bio-Rad One-Step RT-ddPCR Advanced Kit for Probes (cat# 186-4021).
Nested PCR. To amplify single genome FL env, DNA extracted from antemortem PBMCs and post-mortem tissues was diluted using ddPCR quantification data. This limited dilution PCR reaction can prevent PCR recombination and ambiguous base...,

研究目标：鉴定宿主体内HIV在不同身体组织间迁移的系统级特征，并评估这些特征在不同宿主中的存在情况与强度异质性。
研究方法：利用8名HIV感染者的多组织HIV DNA深度测序数据，将组织间HIV迁移的复杂依赖关系建模为网络，并采用指数随机图模型（exponential random graph models, ERGMs）家族对这些网络进行建模。指数随机图模型可用于统计评估网络特征在病毒迁移网络中的出现频率是否显著高于或低于随机预期水平。本分析共考察病毒迁移网络的5项潜在特征：(1) 组织间的双向流动；(2) 同一生物系统内组织间的偏好性迁移；(3) 与HIV储藏库规模相关的病毒迁移水平异质性；(4) 迁移的层级结构；(5) 多组织间的周期性迁移。我们将计算Cochran's Q统计量[原文未完成]。血液与组织样本通过快速尸检流程采集。
基因组DNA提取：从500万外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）以及速冻组织中提取基因组DNA，实验采用QIAamp DNA Mini Kit（Qiagen，货号51306），严格遵循制造商提供的实验流程。提取完成后，通过沉淀法对DNA进行浓缩。
RNA提取：从血浆中分离RNA时，先将500-700μL血浆铺于200μL 20%无菌过滤蔗糖溶液上层，随后以23500×g、4℃条件离心1小时，使病毒颗粒沉淀。弃去上清液，将沉淀重悬于140μL磷酸盐缓冲液（PBS）中。采用Qiagen的QIAamp Viral RNA Mini Kit（货号52904），按照制造商推荐的实验流程完成RNA提取。使用Bio-Rad的One-Step RT-ddPCR Advanced Kit for Probes（货号186-4021），以HIV RNA为模板合成cDNA。
巢式PCR：为扩增单基因组全长env序列，根据液滴数字PCR（droplet digital PCR, ddPCR）的定量结果，对死前采集的PBMCs以及死后组织的提取DNA进行梯度稀释。该有限稀释PCR反应可有效避免PCR重组与碱基识别歧义[原文未完成]。