A DNA primer complementing the template DNA (the DNA to be sequenced) is used in Sanger sequencing to be a starting point for DNA synthesis. In the presence of the four deoxynucleotide triphosphates (dNTPs: A, G, C, and T), the polymerase expands the primordial by adding the additional dNTP to the DNA strand of the template. Four dideoxynucleotide triphosphates (ddNTPs: ddATP, ddGTP, ddCTP, and ddTTP) labeled with a distinct fluorescent dye are used to terminate the synthesis process to establish which nucleotide is incorporated in the nucleotide chain. Compared to dNTPs, ddNTPs has a removed oxygen atom from the ribonucleotide, so it can not form a connection to the next nucleotide. Upon synthesis, the reaction products are loaded into four lanes of a single gel, depending on the different chain-terminating nucleotide and are subjected to gel electrophoresis. Thus the DNA sequence is determined according to their sizes.
Providing creative, accessible and effective solutions for virtually every genomic challenge our customers and partners can face, providing the widest range of sequencing services available today, including applications in human, plant and animal, and microbial research. Our facilities include the latest next-generation sequencing technologies and systems for quality control in the industry. Next-generation sequencing (NGS), also known as high-throughput sequencing, used to describe a number of different modern sequencing technologies. These technologies allow for sequencing of DNA and RNA much more quicker and cheaper than the existing technique like Sanger sequencing, and as such revolutionised the study of genomics and molecular biology.
Whole genome sequencing is the determination of the full DNA sequence of the genome of an organism at a single time , which includes both chromosomal DNA and the DNA found in mitochondria and chloroplasts. De novo sequencing refers to sequencing of a novel genome with no available reference sequence. De novo assembly coverage quality is dependent on the size and continuity of the contigs. De novo sequencing generates a species' first genome map, thus providing a valuable sequence of reference for re-sequencing.
RNA-Seq is the leading mapping and quantification tool for transcriptomes using Next Generation Sequencing ( NGS) technology. The transcriptome refers to the complete collection of transcripts in a cell which provides transcript level information for a specific stage of development or physiological condition. It is important to understand the transcriptome to interpret the functional elements of the genome, and to understand the development and disease. The main purpose of transcriptomics involves cataloging all transcript species; determining the transcriptional gene structure; and quantifying each transcript's expression levels under different conditions.
Small RNA species generally include the most common and well-studied microRNA (miRNA), small interfering RNA (siRNA), and piwi-interacting RNA (piRNA), as well as other types of small RNA, such as small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA). Small RNA is a type of low-abundant, short-length (< 200nt), non-protein-coding, polyadenylated RNAs. Small populations of RNA can vary significantly between various types and species of tissues. Small RNAs are generally formed by fragmentation of longer RNA sequences, using dedicated sets of enzymes and other proteins.
There are huge differences exist between microorganisms and higher eukaryotes. In addition to its smaller genome, most bacteria have a single circular chromosome (sometimes more than one chromosome, linear chromosomes or linear and circular chromosome combinations). The genes in the microbial genomes are typically a single continuous stretch of DNA, while bacterial genome occasionally includes many forms of introns. A further significant distinction is the presence of plasmids in the bacterial genome. Plasmids, the extra-chromosomal circular DNA, can be transferred via horizontal DNA transfer, which mediates the rapid evolution of microorganisms. Virus is an infectious non-cellular organism consisting of a core of DNA or RNA surrounded by a protein coat.
Microbial sequencing of the entire genome yields tons of data allowing a comprehensive evaluation of all the genetic characteristics of an isolated microorganism. Shotgun sequencing strategy is a primary method of sequencing entire microbial genomes. The sequencing steps do not require labor-intensive mapping and cloning, saving huge time and resources. In addition, high-throughput sequencing allows us to sequence hundreds of bacteria or viruses with the power of multiplexing simultaneously. In shotgun sequencing of the whole genome, the whole genome is broken up into tiny fragments for sequencing, and then placed together by statistical methods based on the overlapping regions, thereby having no reference genome.
Single Molecular Real-Time (SMRT) sequencing employs a specialized flow cell with several thousand individual picolitre wells with transparent bottoms — zero-mode waveguides (ZMW). The polymerase is fixed to the bottom of the well, allowing the DNA strand to advance through the ZMW. As a result, a single molecular could be the focus of the system. SMRT sequencing enables real-time imagery of fluorescently tagged nucleotides that are synthesized along with individual molecules of DNA templates. When the template and polymerase dissociate, the sequencing reaction terminates. The PacBio instrument 's average read length is about 2 kb, and some reads may be more than 20 kb. With de novo assemblies of novel genomes which can cover several more repeats and bases, longer reads are particularly useful.
The nanopore based DNA and RNA sequencing technology was developed by Oxford Nanopore Technologies. The Nanopore sequencer is known to be compatible with a range of input materials, including genomic DNA, DNA amplified, cDNA, and RNA. Nanopore biomolecular sequencing technology has wide applications in life sciences, including pathogens detection, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and bacterial antibiotic resistance characterization.
Single nucleotide polymorphisms ( SNPs) are bi-allelic (usually) nucleotide variants occurring at a frequency of around one in 1,000 bp across the genome. They can be present in genome coding, non-coding, and intronic regions, and can influence gene and transcript level transcription factor binding, gene splicing, protein folding, and many other components. Therefore they may be responsible for the variation between individuals and the evolution of genomes, and they are ideal markers for identifying genes associated with complex diseases. SNPs are among the two most common causes of genetic variation (the second is variants of copy number, CNVs). SNP research for studies such as disease-related genetics, individualized health management, reproduction, etc. is essential and very significant.
Human genome comprises approximately 3 X 109 bases, and includes approximately 180,000 (exome) coding regions, comprising approximately 1.7% of a human genome. 85 per cent of the disease-causing mutations are known to occur in the exome. For this reason, the sequencing of the entire exome has the potential to uncover a much lower cost of higher yield of relevant variants than the sequencing of the entire genome. Whole exome sequencing is thought to be an efficient and powerful way to identify the genetic variants that affect heritable phenotypes including significant disease-causing mutations and natural variations that can be used to improve crops and livestock.