RNA sequencing (RNA-Seq) of lymph node, spleen, and thymus transcriptome from wild Peninsular Malaysian cynomolgus macaque (Macaca fascicularis)

Abstract

The cynomolgus macaque (Macaca fascicularis) is an extensively utilised nonhuman primate model for biomedical research due to its biological, behavioural, and genetic similarities to humans. Genomic information of cynomolgus macaque is vital for research in various fields; however, there is presently a shortage of genomic information on the Malaysian cynomolgus macaque. This study aimed to sequence, assemble, annotate, and profile the Peninsular Malaysian cynomolgus macaque transcriptome derived from three tissues (lymph node, spleen, and thymus) using RNA sequencing (RNA-Seq) technology. A total of 174,208,078 paired end 70 base pair sequencing reads were obtained from the Illumina Hi-Seq 2500 sequencer. The overall mapping percentage of the sequencing reads to the M. fascicularis reference genome ranged from 53–63%. Categorisation of expressed genes to Gene Ontology (GO) and KEGG pathway categories revealed that GO terms with the highest number of associated expressed genes include Cellular process, Catalytic activity, and Cell part, while for pathway categorisation, the majority of expressed genes in lymph node, spleen, and thymus fall under the Global overview and maps pathway category, while 266, 221, and 138 genes from lymph node, spleen, and thymus were respectively enriched in the Immune system category. Enriched Immune system pathways include Platelet activation pathway, Antigen processing and presentation, B cell receptor signalling pathway, and Intestinal immune network for IgA production. Differential gene expression analysis among the three tissues revealed 574 differentially expressed genes (DEG) between lymph and spleen, 5402 DEGs between lymph and thymus, and 7008 DEGs between spleen and thymus. Venn diagram analysis of expressed genes revealed a total of 2,630, 253, and 279 tissue-specific genes respectively for lymph node, spleen, and thymus tissues. This is the first time the lymph node, spleen, and thymus transcriptome of the Peninsular Malaysian cynomolgus macaque have been sequenced via RNA-Seq. Novel transcriptomic data will further enrich the present M. fascicularis genomic database and provide future research potentials, including novel transcript discovery, comparative studies, and molecular markers development.

RNA sequencing of kidney and liver transcriptome obtained from wild cynomolgus macaque (Macaca fascicularis) originating from Peninsular Malaysia

“…Preparation of M. fascicularis kidney and liver RNA-seq libraries followed methods previously described in Ee Uli et al [4]. Raw sequencing reads were submitted to NCBI Short Read Archive under accessions SRX2499144-SRX2499147.…”

“…Raw sequencing reads were submitted to NCBI Short Read Archive under accessions SRX2499144-SRX2499147. For data analyses, we used the same bioinformatic workflow and tools as depicted in our earlier publication [4]. Briefly, the assembly of raw sequence reads, normalisation, empirical analysis of differentially expressed genes (EDGE test), and gene ontology (GO) and KEGG pathway annotations were performed in CLC Genomics Workbench (CLCGW) version 7.5.1 (Qiagen Denmark), while functional classification of expressed genes in the kidney and liver transcriptomes was performed using Panther Database version 11.1 released on 2016-10-24 (http://pantherdb.org/) and DAVID Bioinformatics Resources version 6.8 (https://david-d.ncifcrf.gov/home.jsp).…”

“…Briefly, the assembly of raw sequence reads, normalisation, empirical analysis of differentially expressed genes (EDGE test), and gene ontology (GO) and KEGG pathway annotations were performed in CLC Genomics Workbench (CLCGW) version 7.5.1 (Qiagen Denmark), while functional classification of expressed genes in the kidney and liver transcriptomes was performed using Panther Database version 11.1 released on 2016-10-24 (http://pantherdb.org/) and DAVID Bioinformatics Resources version 6.8 (https://david-d.ncifcrf.gov/home.jsp). To better present the tissue-specific genes, we combined gene expression data from this study and our previous study [4] to create a Venn diagram representation of tissue-specific genes using the web tool Venn Diagrams (http://bioinformatics.psb.ugent.be/webtools/Venn). Validation of the RNA-seq dataset was performed with NanoString nCounter Elements XT (NanoString Technologies Inc., Seattle, WA, USA) gene expression assay.…”

“…Validation of the RNA-seq dataset was performed with NanoString nCounter Elements XT (NanoString Technologies Inc., Seattle, WA, USA) gene expression assay. The methodology was the same as previously described in Ee Uli et al [4]. Additional file 1 lists the genes utilised for the RNA-seq validation and their respective probe pair sequences.…”

“…This study describes the RNA sequencing (RNA-seq) of two tissues—kidney and liver—harvested from wild Peninsular Malaysian cynomolgus macaques, and is an extension of a previous study [4] whereby the lymph node, spleen, and thymus transcriptomes of wild Peninsular Malaysian M. fascicularis were also sequenced with RNA-seq technology. An additional 75,350,240 sequence reads were obtained from this study, supplementing the previous wild Peninsular Malaysian M. fascicularis dataset for downstream applications.…”