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| Status |
Public on Nov 01, 2016 |
| Title |
Next Generation Sequencing Facilitates Quantitative Analysis of HIV-1 Latency in Central Memory T Cells |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Purpose: Next-generation sequencing (NGS) has become a powerful microscope to study cell models of HIV. The goals of this study is to analyze latent HIV infection in the TCM model of HIV latency described in Martins et al 2015 and to evaluate potential markers for HIV infection.
Methods: Peripheral blood mononuclear cells were collected from 4 healthy donors. Naive CD4 T cells were isolated and utilized to generate a model of latent HIV infection. During model generation, cells were sorted and collected for RNA extraction 10 days post infection along with their uninfected counterparts. 2 days after this, cells were activated with CD3/CD28 stimulation and collected for study for a total of 16 samples from 4 donors. After cell collection, RNA was extracted from infected cells and their uninfected counterparts for deep sequencing by Expression Analysis. Sequence reads that passed quality filters were mapped using Tophat and counted using HTSeq. In addition to human transcripts, we utilized 92 ERCC spikes as negative controls. Any human gene which did not achieve at least 1 count per million reads in at least 4 samples or any ERCC that did not achieve at least 5 reads in at least 4 samples was discarded. Differential expression for activated samples was not performed, but rather used to demonstrate upregulation of T-cell activation markers along with changing to the type and abundance of HIV transcripts produced.
Results: Using an custom built data analysis pipeline, 82 million reads per sample to the human genome (build hg38) and identified 13,534 human transcripts 67 ERCC spike in transcripts, and HIV NL43 transcripts were identified with the Tophat/HTSeq workflow. Differential expression analysis was performed between uninfected and latently infected cells. 826 genes were found to be differentially expressed, 275 downregulated and 551 upregulated (FDR < 0.05) with EdgeR. GO and Pathway analysis of differential expressed genes revealed significant dysregulation of genes involved in the p53 signaling pathway. Subsequent studies with pifithrin demonstrated a reduction in latently infected cell during model generation.
Conclusions: This study represents the first detailed analysis of HIV latency with this cell model using next generations sequencing. These results demonstrate that the TCM model of HIV latency of Martins et al 2015 is truly reflective of HIV latency. This study also provides a framework for which to analyze future cell models of HIV latency using next generation sequencing. Finally, this work demonstrates that the p53 signaling pathway is a key pathway dysregulated in latency for this cell model with several genes dysregulated in HIV latency.
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| Overall design |
TCM model of HIV latency mRNA profiles of 4 donors in 4 conditions were generated by deep sequencing by Expression Analysis.
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| Contributor(s) |
White CH, Moesker B, Beliakova-Bethell N, Martins L, Spina CA, Margolis DM, Richman DD, Planelles V, Bosque A, Woelk CH |
| Citation(s) |
27898737, 31710657 |
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| Submission date |
May 24, 2016 |
| Last update date |
Nov 25, 2019 |
| Contact name |
Cory Haley White |
| Organization name |
Merck
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| Department |
DAGS
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| Lab |
IDeA Lab
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| Street address |
320 Bent St
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02141 |
| Country |
USA |
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| Platforms (1) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
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| Samples (16)
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| Relations |
| BioProject |
PRJNA322599 |
| SRA |
SRP075608 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE81810_RAW.tar |
3.3 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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