The Genome Analysis Core

基因组分析核心（TGAC）成立于2014年，隶属于人类免疫学科，旨在满足疫苗研究中心（VRC）的许多专业测序需求。目前，VRC正在设计、生产和测试候选免疫原，旨在激发针对艾滋病毒和其他传染性病原体的广泛中和抗体。这项工作的核心是需要诱导抗体，这些抗体使用特定的IgH和IgL链基因对，这些基因对经历一系列连续的体细胞超突变（SHM）来实现有效的功能。为了评估候选疫苗，需要使用各种分子技术监测这些抗体的演变，从而监测疫苗的有效性，这些技术最终达到对特定B细胞Ig基因进行单细胞NGS的目的。此外，被动抗体的应用必须依赖于hiv感染者中广泛中和的env特异性抗体的鉴定和测序。TGAC开发了许多下一代测序方法。其中包括：用于无偏倚TCR和Ig测序的散装和单细胞5RACE，用于Ig位点靶向测序的基因组诱饵，用于人、小鼠和NHP的散装mRNAseq和单细胞mRNAseq。此外，已经开发了许多HIV测序方法，我们已经使用这些方法来表征感染者体内的HIV库，作为治疗研究的一部分，并在被动给药env特异性bnAbs的临床研究中监测逃逸变体的选择。我们已经购买了两个10倍的Genomic Chromium平台，我们目前正在使用这些平台来提高单细胞5和3转录组测序的吞吐量。我们已经成功开发并实施了一种定制引物库准备方法，因此我们可以将标准的10x5免疫球蛋白和T细胞受体测序方案用于恒河猴、食蟹猕猴和豚鼠的样本。此外，我们还将库准备的灵敏度提高了100倍。我们已经实现了使用自定义偶联抗体的CITE-seq应用程序来执行高参数细胞表面蛋白质组学。此外，VRC正在进行的许多其他研究项目，如流感、呼吸道合胞病毒、疟疾和结核病疫苗的研究项目，都需要如上所述的Ig、TCR和转录组测序。最后，我们有一个强大的微生物组测序程序。

The Genome Analysis Core (TGAC), within the Human Immunology Section, was set up in 2014 to fulfill many of the specialist sequencing needs of the Vaccine Research Center (VRC). Currently, the VRC is designing, producing and testing candidate immunogens aimed at eliciting broadly neutralizing antibodies against HIV and other infectious agents. At the core of this effort lies the requirement to elicit antibodies that use particular IgH and IgL chain gene pairs which undergo a sequential series of somatic hypermutations (SHM) to achieve effective function. For evaluation of vaccine candidates, the evolution of such antibodies, and thus the effectiveness of the vaccine, needs to be monitored using a variety of molecular techniques that culminate in single-cell NGS of specific B cell Ig genes. In addition, passive antibody applications necessarily depend on the identification and sequencing of broadly neutralizing Env-specific antibodies in HIV-infected people. TGAC has developed a number of next-generation sequencing methodologies. These include: bulk and single cell 5RACE for unbiased TCR and Ig sequencing, genomic baiting for targeted sequencing of Ig loci, bulk mRNAseq and single cell mRNAseq for human, mouse and NHP. In addition, a number of approaches for sequencing HIV have been developed which we have used to characterize the HIV reservoir in infected people, as part of cure studies, and also to monitor the selection of escape variants in clinical studies of passive administration of Env-specific bnAbs. We have purchased two 10x Genomic Chromium platforms and we are currently using these to increase our throughput of single cell 5 and 3 transcriptome sequencing. We have successfully developed and implemented a custom primer library prep approach so that we may adapt the standard 10x 5 immunoglobulin and T cell receptor sequencing protocol for use with samples from rhesus and cynomolgus macaques, and Guinea pigs. In addition, we have increased the sensitivity of the library prep by 100-fold. We have implemented CITE-seq applications using custom conjugated antibodies to perform high parameter cell surface proteomics. Furthermore, many other ongoing research projects at the VRC such as those for influenza, RSV, malaria and TB vaccines require Ig, TCR and transcriptome sequencing as described above. Finally, we have a robust microbiome sequencing program.