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| Status |
Public on Mar 14, 2019 |
| Title |
E07B-3059241-12C44-TimePoint02-BoneMarrow-16 |
| Sample type |
SRA |
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| Source name |
Bone Marrow
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| Organisms |
Plasmodium knowlesi; Macaca fascicularis |
| Characteristics |
collection time point (#) or necropsy: 2
gender: Male
non human primate individual id: 12C44
tissue: Bone Marrow
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| Treatment protocol |
The E07 cohort was inoculated with freshly dissected P. knowlesi sporozoites on 11/01/16. However, for unexplained reasons blood-stage parasitemias did not occur. Consequently, the E07 cohort was reinoculated with cryopreserved P. knowlesi sporozoites. This inoculation was performed on 1/20/17. Hence 'E07A' refers to samples and results from the failed inoculation from 11/01/16 and 'E07B' refers to samples and results from the successful inoculation on 1/20/17). Telemetry devices (DSI, model L11) with blood pressure sensors and electrocardiogram (ECG) leads were surgically implanted in seven malaria-naive male long-tailed macaques (Macaca fascicularis), approximately five years of age. After a resting period of three weeks, the telemetry implants were turned on and physiological data that include activity, temperature, ECG, and blood pressure were continuously collected. After the E07A failed infection using a fresh preparation of salivary gland sporozoites, the implants were deactivated to preserve battery life. Between E07A and E07B, a single rhesus macaque (M. mulatta) was added to the cohort as an infection control with no telemetry implant. At the start of E07B, telemetry implants were reactivated. Ten days after reactivation all animals were inoculated intravenously with cryopreserved P. knowlesi Malayan strain salivary gland sporozoites, obtained from Anopheles dirus infected with parasites from the Pk1A+ clone and previously tested in E30 for their infectivity of macaques. The sporozoite stocks used were produced, isolated and cryopreserved at the Centers for Disease Control and Prevention, and then stored at Yerkes. After inoculation, the macaques were profiled for clinical, hematological, parasitological, immunological, functional genomic, proteomic, and metabolomic measurements. The experiment was designed with pathology studies and thus terminal necropsies, which were scheduled at the log phase of the infection, at the peak of parasitemias, at the middle of the chronic phase, or at the end of the follow-up period of 45 days after the inoculation of the sporozoites. Capillary blood samples were collected daily for the measurement of complete blood counts (CBCs), reticulocytes, and parasitemias. Capillary blood samples were collected every other day to obtain plasma for metabolomic analysis. Venous blood and bone marrow samples were collected at six time points for functional genomic, targeted proteomic, targeted metabolomics, and immunological analyses. Physiological data noted above were continuously captured via telemetry. Within the MaHPIC, this project is known as ‘Experiment 07 (E07A and 07B)’. This dataset was produced by Dr. Steven E. Bosinger, Nirav Patel, and Gregory K Tharp at the Emory University Yerkes Genomics Core. To access other publicly available results from ‘E07' and other MaHPIC Experiments, including clinical results (specifics on drugs administered, diet, and veterinary interventions), and other omics, visit http://plasmodb.org/plasmo/mahpic.jsp . This page will be updated as datasets are released to the public. The experimental design and protocols for this study were approved by the Emory University Institutional Animal Care and Use Committee (IACUC) and the MRMC Office of Research Protection Animal Care and Use Review Office (ACURO). This dataset contains results from both E07A and E07B. During the experiment subjects experienced chronic infection. Samples were collected either daily for select specimen types or on clinically determiend time point days. Time points were planned based on the expected progression of infection. Sample collection at time points was planned to coincide with this expected clinical progression. However, because of individual variations in the clinical progression of infection for each NHP the clinically defined stage of infection may differ between NHPs at the same time point. For this reason it is imortant for anyone consuming these data to be aware of any differences between the 'Idealized' and 'Actual' disease progression for each NHP and the corresponding clinical stage of each NHP at each time point when samples were collected. For this reason the supplementary file E07M99MEMfKnXXZZ_Supporting-Clinical-Information_MULTIPL.pdf is provided to clarify both the 'Idealized' or planned collection of time point samples and the clinically-defined 'Actual' disease progression for each NHP. Anyone consuming these data should use this information to ensure they are aware of the clinical state of each NHP at each time point and be aware of differences between NHPs. [E07A] Time point 1 = Pre-telemetry surgery implants baseline - NHPs were not yet infected with P. knowlesi. This time point is prior to failed inoculation with fresh sporozoites. [E07A] Time point 2 = Post-telemetry surgery baseline - NHPs were not yet infected with P. knowlesi. This time point is prior to failed inoculation with fresh sporozoites. [E07A] Time point 3 = Post-failed E07A inoculation - Post failed inoculation with fresh sporozoites, three days after failed inoculation. Intended to take place during liver stage infection. [E07B] Time point 2 = Baseline 2 post-telemetry reactivation - Post-telemetry implantation baseline control: TP2 occurred after telemetry implant reactivation following E07A. NHPs were not yet infected with P. knowlesi. [E07B] Time point 3 = Pre-Patent / Early Infection - Post-inoculation with cryopreserved sporozoites: TP3 took place three days after inoculation and is representative of the liver stage infection. [E07B] Time point 4 = Log Phase - Log phase / pre-peak phase: This time point was intended to examine responses prior to the peak of parasitemia. Activation at cellular and immune levels, as well as differential telemetry results compared to baseline readings, was expected. Parasitemia of ~100-10,000/ul was expected. [E07B] Time point 5 = Peak/Post-Peak of Infection - Peak/post-peak infection: This time point examined the response of the immune system and metabolism of the host during the peak of infection whenever the animal infected with P. knowlesi is experiencing clinical signs of disease. Post-peak was observed for 12C53 and 11C131. [E07B] Time point 6 = Chronic Phase 1 - Chronic phase of infection: M. fascicularis infected with P. knowlesi can self-control hyper-parasitemias, and they were expected to reach a chronic phase 20-25 days after experimental infection whenever the animal is experiencing clinical signs of disease. This time point examined the response of the immune system and metabolism of the host in early stages of the chronic infection. [E07B] Time point 7 = Chronic Phase 2 - Chronic phase of infection: M. fascicularis infected with P. knowlesi can self-control hyper-parasitemias, and they were expected to reach a chronic phase 20-25 days after experimental infection whenever the animal is experiencing clinical signs of disease. This time point examined the response of the immune system and metabolism of the host in late stages of the chronic infection. E07B] Necropsy = Necropsy - Terminal endpoint: Staggered necropsy dates were carried out for all NHPs (days 10, 14, 27 or 45). The final necropsy occurred 45 days after experimental infection.
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| Growth protocol |
Animals approved for use were moved into experimental housing 10 days prior to the start of the experiment.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Please note that the RNASeq data for this experiment was produced across three specimen types (venous whole blood (WB), bone marrow (BM), and spleen tissue (SP)) using RNAlater and PAXgene sample collection and storage technologies. Please carefully review the two README files provided as supplementary files with this dataset for the details on the methods used for sequence generation and abundance measures. README file names are as follows: E07M99YSMfKnXXZZ_06062018-Readme_MULTIPL_GEO.txt E07M99FGMfKnXXZZ_06292018-Readme_MULTIPL_GEO.txt
Approximately 1 μg of total RNA per sample was converted to double-stranded cDNA using poly-A beads to enrich for mRNA, and Illumina TruSeq Stranded mRNA Sample Prep kits to generate strand-specific libraries. As a quality control, 92 spike-in RNAs of known concentration and GC proportions (ERCC Spike-In Control, Life Technologies) were added to constitute approximately 1% of the total RNA for each library. Adapters were ligated to facilitate multiplexed sequencing on an Illumina HiSeq3000 at the Yerkes Genomics Core, aiming for 50 million paired-end 100 base pair (bp) reads per library.
RNA was extracted using Tempus-Spin RNA isolation kits. The quality of all RNA samples was confirmed using a Bioanalyzer, with an RNA Integrity Number (RIN) greater than 8 recorded for all samples."
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
Please note that the RNASeq data for this experiment was produced across three specimen types (venous whole blood (WB), bone marrow (BM), and spleen tissue (SP)) using RNAlater and PAXgene sample collection and storage technologies. Please carefully review the two README files provided as supplementary files with this dataset for the details on the methods used for sequence generation and abundance measures. README file names are as follows: E07M99YSMfKnXXZZ_06062018-Readme_MULTIPL_GEO.txt E07M99FGMfKnXXZZ_06292018-Readme_MULTIPL_GEO.txt Please note that even though all file names indicate that the host is Macaca fascicularis, some (from NHP RAw8) are derived from M. mulatta.
E07M99FGMfKnXXBM_Fascicularis-Genes-RawCounts-Results_MULTIPL_GEO.xlsx, E07M99FGMfKnXXBM_Knowlesi-Genes-RawCounts-Results_MULTIPL_GEO.xlsx
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| Data processing |
Bases were called with Illumina RTA (Real-Time Analysis, v2.7.7) with default parameters. Illumina bcl2fastq v2.17.1.14 was used for demultiplexing. FASTQC (v0.10.1) was used to assess data quality, but the data were not filtered at this stage.
Reads were mapped to a composite reference assembly consisting of host, parasite, and ERCC control references with STAR (v2.5.2b) with default alignment parameters.
Abundance estimation of raw read counts per transcript was done internally with STAR using the algorithm of htseq-count.
Genome_build: RNA-Seq reads were mapped to both host and parasite genomes. Host (Mf): The version of the Macaca fascicularis used was obtained from ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/364/345/GCF_000364345.1_Macaca_fascicularis_5.0 . For more information see https://www.ncbi.nlm.nih.gov/assembly/GCF_000364345.1/ . Parasite: Plasmodium knowlesi PK1 (A+) Malayan H strain genome assembly and annotation was used. The assembly has been deposited in GenBank under the BioProject accession PRJNA377737 Note that M. mulatta samples were also mapped to the M. fascicularis reference genome.
Supplementary_files_format_and_content: Excel files contain raw counts at the gene level, for each individual. Raw counts are further classified by experimental Time Point and Specimen Type. Note that in addition to raw counts, normalized data are available as supplementary files as described in the supplementary file E06M99FGMmKnXXZZ_06292018-Readme_MULTIPL_GEO.txt.
Supplementary_files_format_and_content: Host and Parasite Raw Read Counts Column headers are defined as follows: 'Gene ID': Identifiers of all Genes in the annotation. 'Gene Symbol': Symbols of all Genes in the annotation. 'Sample Identifier / Raw file list / Abundances': The raw file names and sample identifers both contain information regarding Specimen Type, NHP ID, and Time Point. Abundances measures are from the raw files listed. Example Notes: There are 12 columns representing 2 animals for Time Points T01-T05 and Necropsy. There is a read count entry per sample for every gene that appears in the annotation. For genes where there was no detection of expression by reads that mapped to their loci, the raw count is 0.
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| Submission date |
Mar 11, 2019 |
| Last update date |
Mar 14, 2019 |
| Contact name |
Mary Galinski |
| Organization name |
Emory University
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| Department |
Vaccine Center at Yerkes
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| Lab |
Galinski Lab
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| Street address |
954 Gatewood Road
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| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30329 |
| Country |
USA |
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| Platform ID |
GPL26280 |
| Series (2) |
| GSE94274 |
An Integrated Approach to Understanding Host-Pathogen Interactions |
| GSE128115 |
Malaria Host Pathogen Center Experiment 07A and 07B - Macaca fascicularis infected with Plasmodium knowlesi to produce and integrate clinical, hematological, parasitological, omics, telemetric and histopathological measures of acute primary infection. |
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| Relations |
| BioSample |
SAMN11099895 |
| SRA |
SRX5503964 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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