DNA sequencing is a fundamental genomic technology that determines the precise order of nucleotides in DNA molecules, enabling comprehensive analysis of genetic variations, gene expression patterns, and molecular mechanisms underlying disease. This powerful tool has revolutionized preclinical research by providing unprecedented insights into genome structure, function, and variation across animal models and experimental systems in contract research settings.
Technology Evolution and Next-Generation Sequencing
The onset of DNA sequencing dates back to the 1970s when Fred Sanger developed a sequencing method based on polynucleotide chain termination, earning him recognition as the father of sequencing. Next-generation sequencing (NGS) has revolutionized nearly every area of biotechnology and has been applied to various aspects of biological science, including animal, human, and plant biotechnology.
NGS is a massively parallel sequencing technology that offers ultra-high throughput, scalability, and speed, used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA. Using capillary electrophoresis-based Sanger sequencing, the Human Genome Project took over 10 years and cost nearly $3 billion, while next-generation sequencing makes large-scale whole-genome sequencing accessible and practical.
Whole Genome Sequencing Applications
Whole-genome sequencing (WGS) is a powerful and comprehensive genomic analysis technique that involves determining the complete DNA sequence of an individual’s genome, providing a detailed blueprint of genetic makeup. It finds application mainly in discovery science, such as plant and animal research, cancer research, rare genetic diseases, patients with complex disease symptoms, population genetics, and novel genome assembly.
Advancements in massively parallel short-read sequencing technologies and associated decreasing costs have led to large and diverse variant discovery efforts across species. WGS projects may range from single individuals with phenotypes of interest to entire cohorts, broadening ability to answer biological questions at population-level scales.
Exome Sequencing and Targeted Approaches
Exons are the genome’s protein-coding regions and are collectively known as the exome, making up approximately 2% of the whole genome but encoding most known disease-related variants. Because most known mutations that cause disease occur in exons, whole exome sequencing is thought to be an efficient method to identify possible disease-causing mutations.
Whole exome sequencing allows variations in protein-coding regions of any gene to be identified rather than in only select few genes. However, researchers have found that DNA variations outside exons can affect gene activity and protein production and lead to genetic disorders—variations that whole exome sequencing would miss.
RNA Sequencing and Transcriptome Analysis
RNA sequencing (RNA-seq) is a genomic approach for detection and quantitative analysis of messenger RNA molecules in biological samples and is useful for studying cellular responses. Recently, RNA-Seq technology has increasingly been used in livestock, wild and aquatic animal studies to measure gene expression analysis and SNP discovery.
Whole Transcriptome Sequencing allows characterization of all types of RNA transcripts (coding and non-coding RNAs) of particular organisms, irrespective of whether they are polyadenylated. Single-cell RNA-sequencing (scRNA-seq) has enabled researchers to examine the fundamental unit of biology—the cell—since the first study was published in 2009.
Animal Research and Preclinical Applications
The enormous information produced by NGS assists in understanding genomic variations, disease mechanisms, and resistance, thus helping development of better diagnostics, therapies, and breeds. A study of specific species adaptation using DNA sequencing was performed on painted turtles, revealing genetic mechanisms responsible for extraordinary ability to survive without oxygen for several months.
Preclinical development is grappling with physiological matters of increasing intricacy—polygenicity, genetic diversity, microbiome composition—requiring more human-like animal models. Animal model providers are taking advantage of next-generation sequencing technology, genome engineering tools, and advanced computational approaches including big data analytics and machine learning.
Variant Discovery and Genomic Analysis
Whole Animal Genome Sequencing (WAGS) is an open-source set of user-friendly, containerized pipelines designed to simplify the process of identifying germline short (SNP and indel) and structural variants geared toward the veterinary community. Although only 40% of exonic variants identified by whole genome sequencing were captured using RNA-Seq, this number rose to 81% when concentrating on genes known to be well-expressed in source tissue.
Three-platform sequencing (whole-genome, whole-exome, and whole-transcriptome) has positive predictive values of 97-99%, 99%, and 91% for somatic SNVs, indels and structural variations respectively, based on independent experimental verification. This comprehensive approach enables detection of diverse classes of somatic and germline mutations relevant to research applications.
Clinical and Diagnostic Applications
Investment in development of NGS technologies was made with the goal of expediting use of genome sequencing data in clinical practice of medicine. Preclinical applications of NGS include all experiments that characterize genetic and epigenetic profiles of disease states to enhance understanding of molecular basis for disease pathogenesis.
The Virology Laboratory offers NGS services for viral diagnostics and whole genome sequencing, including exploratory metagenomic sequencing to detect viruses in a wide variety of species and sample types. These services enable comprehensive viral characterization and surveillance across multiple animal species.
Technology Platforms and Methodologies
The Illumina platform uses bridge amplification for polony generation and sequencing by synthesis, with major advantages being relatively inexpensive price per base and comparatively high sequencing depth. The Ion Torrent technology platform uses sequencing by synthesis strategy but directly detects hydrogen ions when bases are incorporated into the growing strand.
A deep learning model for predicting Next-Generation Sequencing depth from DNA probe sequences includes bidirectional recurrent neural networks that take as input both DNA nucleotide identities and calculated probability of nucleotides being unpaired. This computational approach addresses non-uniform coverage challenges in targeted sequencing applications.
Data Analysis and Bioinformatics
Two major challenges faced with RNA-Seq are differences in sequencing technologies and bioinformatics analysis. This groundbreaking technology has enabled extensive research and allowed scientists to explore complexities of genetic information in unprecedented ways, with high-throughput capacity and cost-effectiveness.
Clinical cancer genomic profiling by three-platform sequencing achieves high accuracy by cross-validating variants between sequencing types, thereby removing need for confirmatory testing and facilitating comprehensive reporting in clinically-relevant timeframes.
Global Impact and Future Directions
In less than 25 years, animal genome science has transformed from a discipline seeking first glimpses into genome sequences across the Tree of Life to a global enterprise with ambitions to sequence all Earth’s eukaryotic biodiversity. Animal genome assemblies have been contributed by researchers at institutions on every continent with permanent inhabitants, including 52 countries.
Since 2005, advances in next-generation sequencing technologies have revolutionized biological science, with analysis of environmental DNA through specific gene markers being a key application in ecological and environmental research. The technology continues to expand applications in understanding genomic diversity and evolution across species.
Anilocus provides comprehensive DNA sequencing services including whole genome sequencing, exome sequencing, RNA-seq, and targeted sequencing approaches for animal research applications. Our facility offers advanced bioinformatics pipelines, variant discovery analysis, and complete genomic characterization to support preclinical research programs from target identification through therapeutic development. Our sequencing capabilities include single-cell RNA sequencing, epigenomic analysis, and comparative genomics studies with expert data interpretation and publication-ready results.
Contact us for specialized DNA sequencing study design and genomic analysis services.