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Sample GSM5778107

Query DataSets for GSM5778107

Status

Public on Feb 10, 2022

Title

MBH31

Sample type

SRA

Source name

Whole blood

Organism

Macaca mulatta

Characteristics

vaccine administered: Ad26.CoV.S
ad26.cov. dose: 5.00E+10 vp
time post-vaccination: D1

Treatment protocol

20 outbred Indian-origin adult male and female rhesus macaques (Macaca mulatta) ages 6-10 years old were randomly allocated to groups. All animals were housed at Bioqual, Inc. (Rockville, MD). Animals were immunized with 1011 vp (N=10) or 5x1010 vp (N=10) Ad26.COV2.S and were followed for either 230 or 315 days. Animals were then boosted with 5x1010 vp Ad26.COV2.S (N=10) or Ad26.COV2.S.351 (N=10). Half of animals in each original dose group were immunized by each booster vaccine. All immunologic studies were performed blinded. Animal studies were conducted in compliance with all relevant local, state, and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee (IACUC).

Extracted molecule

total RNA

Extraction protocol

RNA was isolated from blood samples stored in Paxgene tubes at the Yerkes National Primate Center for library preparation (http://www.yerkes.emory.edu/nhp_genomics_core/). RNA quality was assessed using an Agilent 4200 TapeStation and concentration via the RNA HS assay on the Qubit. Globin transcripts in the blood RNA were blocked with the FastSelect Globin Reagent (Qiagen) prior to library preparation. Libraries were prepared using the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) in combination with the NexteraXT DNA Library Preparation kit to append dual-indexed adapter sequences (Illumina). Libraries were validated by capillary electrophoresis on an Agilent 4200 TapeStation, pooled at equimolar concentrations, and sequenced on an Illumina NovaSeq6000 at 100SR, yielding 25-30 million reads per sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

p20185-s018_20-17_BH31_D1_S254

Data processing

Raw reads were examined for quality issues using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to ensure library generation and sequencing are suitable for further analysis. Reads were aligned using STAR v2.7.3. Reads were aligned to Macaca mulatta genome . We used the macaques genome reference Macaca mulatta - MacaM (https://www.unmc.edu/rhesusgenechip/index.htm). The counts of reads aligning to annotated genes was obtained from STAR ReadsPerGene files. We used DESeq2 (Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2) internal filtering algorithm called "independentFiltering" that sets a threshold on the mean of normalized counts of all samples (baseMean > 10) in order to maximize the number of tests that pass multiple test correction. DESeq2 was used to generate the normalized read count table based on their estimateSizeFactors() function with default parameters by calculating a pseudo-reference sample of the geometric means for each gene across all samples and then use the "median ratio" of each sample to the pseudo-reference as the sizeFactor for that sample. The sizeFactor is then applied to each gene's raw count to get the normalized count for that gene. Differential expression at the gene level were performed by DESeq2 implemented in the DESeq2 R package, using counts per gene generated by a custom script that pulls out the library prep abundance estimation column into files, and read those files into DESeq2 with the DESeqDataSetFromHTSeqCount() function. A corrected p value cut-off of 0.05 was used to assess significant genes that were upregulated or down regulated at days 2, 7, 14 and 28 compared to baseline using Benjamini-Hochberg (BH) method.
Gene set enrichment analysis and a compendium of databases of biological and immunological pathways were used to test the longitudinal enrichment of pathways on days 2, 7, 14 and 28 post vaccination compared to baseline. Genes were pre-ranked by fold change from the highest to the lowest and GSEA was used to assess the enrichment of selected gene sets. Cytokines signaling, immune cell signatures, and molecular pathways were compiled from the MSigDB Hallmark, C2, C7 and C3 gene sets
(https://www.gsea-msigdb.org/gsea/msigdb/collections.jsp), and the blood transcriptional modules (BTMs) (25). The GSEA Java desktop program was downloaded from the Broad Institute (http://www.broadinstitute.org/gsea/index.jsp) and used with GSEA Pre-Ranked module parameters (number of permutations: 1,000; enrichment statistic: weighted; 10≤ gene set size ≤5,000). Sample-level enrichment analysis (26) was used to investigate the enrichment of pathways in each individual animal. Briefly, the expression of all the genes in a specific pathway was averaged across samples and compared to the average expression of 1,000 randomly generated gene sets of the same size. The resulting Z score was then used to reflect the overall perturbation of each pathway in each individual sample. Pathways SLE scores were correlated with binding and neutralizing titers and B cell responses elicited by Ad26 days following vaccination, using Spearman correlation method.
Pharmacokinetics. To estimate antibody decay rate, the antibody median values for 1x1011 and 5x1010 dose groups were plotted as a function of time post-vaccination, and the data were fitted to a biphasic decaying (27) exponential model using the Curve Fitting Tool in MATLAB (version R_2021a). The half-life of the fast and slow phases of each decay model are indicated on the graphs.
Genome_build: MacaM (https://www.unmc.edu/rhesusgenechip/index.htm)

Submission date

Jan 07, 2022

Last update date

Feb 10, 2022

Contact name

Dan Barouch

Organization name

BIDMC

Department

CVVR

Lab

Barouch Lab