Loan Nguyen

Cas9 targeted enrichment of age-related sites

Tools to predict birthdates of cattle are desperately required by industry to ensure compliance with breed registration requirements and to increase the rate of genetic gain for traits such as growth rate and fertility. This study will use new methods of gene targeting and sequencing to investigate the predictive ability of the methylation status of key genes related to age in mammals. Several studies found age-related-conserved sites among species. From these a list of 43 age related genes in cattle has been derived. In this study these genes will be targeted for sequencing and methylation calling in cattle of varying ages. A predictive statistical approach will then the used to associated the methylation rates of those genes with animal age, which can then be used to calculate birthdate.

This project will apply long-read Oxford Nanopore Sequencing CAS9 targeted enrichment. The project aims to use this approach to target age-related-conversed genes among humans, dogs, and cattle. Finally, validation in large populations will be performed with the most significant age-related sites using quantitative methylation-specific PCR. The ultimate aim of this work is to develop an on farm diagnostic tool that will allows producers to record accurate birthdates and improve the profitability of the beef industry through genetic gain for key traits.

This project will develop skills in bioinformatics and molecular biology. Students will learn how to design experiments, perform sequencing, and manage very large sequence data sets.

A comparison of Full-length transcript sequencings methods

Precise gene annotations are essential to understanding the complexity of a species transcriptome and to connect genomic sequence to gene function and ultimately phenotype. RNA sequencing has been widely applied to build a reference transcriptome using short-read sequencing, followed by the assembly or mapping reads to accessible reference genomes. However, short-read sequencing is facing the challenge of lengthy transcripts, repetitive regions, and transposable elements. Long read-sequencing technology, represented by Pacbio Sequencing and Oxford Nanopore sequencing, has overwhelmed this challenge by generating full-length transcripts. Another advantage of Oxford Nanopore sequencing is the potential to direct sequence RNA molecules to remove PCR bias and identify the base modifications.

In this study, students will perform direct cDNA sequencing and direct RNA sequencing from the liver samples from two cattle genomes using Pacbio isoform sequencing and Nanopore sequencing. The generated sequencing datasets will be compared between technologies and published sequencing data from the same tissues (RNAseq and CAGEseq). The ultimate aim of this project is to advance our understanding of emerging technologies and deeping our understanding of the cattle transcriptome.

This project will developed molecular techniques (DNA extraction, library preparation and sequencing) and bioinformatics skills. Students will learn how to work with RNA samples and manage extensive sequencing data sets.

DNA extraction method for faecal metagenomics to assess cattle diet

Cattle diet history information can be obtained by studying non-invasive samples, like dried faecal samples. Additionally, the analysis of faecal samples can also provide the information about the digestive efficiency of an animal. The advent of improved sequencing methodologies has simplified the characterization of complex faecal DNA and allows for the characterization of diet profiles by matching the faecal sequence data with available sequence databases of potential food sources. In this study, student will employ different extraction protocols to isolate DNA from faecal samples using a variety of molecular techniques in the lab.

Predicting age using methylated sites

In humans, the methylation state of CpG sites changes with age and can therefore be utilized as an accurate biomarker for aging. In cattle, biological age prediction based on methylation status could provide key information for genetic improvement programs. Additionally, comparing chronological age with biological age (based on methylation status) can provide important information about the stress an animal has been under during its lifetime. However, relatively little is known about DNA methylation patterns in cattle. Students will use cutting edge data sources including reduce representation bisulphite sequencing data, whole genome bisulphite sequencing, long read sequencing and human methylation data to identify differentially methylated regions between old and young animals and validate those regions with modern molecular technologies.