Investigation into the Genetic Variation of Toll-Like Receptor 9 in Cattle Using Both Sanger and Next-Generation Sequencing from FTA-Cards
Abstract
The susceptibility or resistance to infectious diseases depends on the host immune response. The genetic variation of immune-relevant genes such as Toll-Like Receptor (TLR) genes is associated with susceptibility or resistance to the pathogen. In this thesis, the genetic variation of Toll-like receptor 9 (TLR9) was investigated; this gene is known to have a function in parasite recognition. In a previous study from the Hide lab, 80 bovine blood samples were collected from bulls in Ahoada, Nigeria and the samples were stored on Whatman FTA cards. In this study bovine genomic DNA was extracted from the FTA cards using three different approaches: 5% Chelex resin, the REPLI-g Mini (Qiagen) and the DNeasy Blood & Tissue Kit (Qiagen). The most efficient method for DNA extraction was via the DNeasy Blood & Tissue (Qiagen) kit and this allowed a PCR protocol to be developed for amplification of a fragment of the bovine TLR9 gene. Subsequently, Sanger sequencing and Next Generation Sequencing (NGS) approaches validated the existence of genetic variation across a 554 bp region of exon 2 (f1TLR9) of the bovine TLR9 gene. Moreover, our data revealed that there is no significant correlation between Trypanosoma infection and the existence of genetic variations of f1TLR9 in Bos. indicus cattle from Nigeria; the interrater reliability between NGS and Sanger sequencing was intermediate. In conclusion, high-quality bovine DNA was extracted from FTA cards to allow PCR amplification and detection of genetic variation within exon 2 of the TLR9 gene by two independent sequencing approaches. These results provide a methodology for facilitating future studies into the existence of genetic variation in other TLR and immune-relevant genes and whether, or not, these correlate with disease susceptibility, or resistance, in both humans and animals.