Bioinformatics: Sequencing Sequencing · Common tool for life scientists . The story echoes that of computing; once computers became fast & cheap, they were adopted everywhere. Human Genome Project · Depended crucially on contributions by computer scientists, especially new methods for assembling DNA fragments into chromosomes. Computational Genomics(continued): · The idea of using high-throughput DNA sequencing in medical settings is only possible because of novel, extremely efficient software developed in the years after second-generation sequencers arrived. Steps for Next Generation Sequencing: 1. Samples are taken from an organism; that sample could be saliva, biopsy, DNA or a tissue. 2. DNA is extracted from cells. 3. DNA is randomly shattered into fragments(shorter pieces); these fragments are usually short(100 to 500 base pairs). 4. Library construction separates DNA templates and each one is sequenced independently. DNA . DNA is composed of two chains(strands) that coil around each other and contain the same genetic information. · DNA has alternating base pairs.
· Any biological substance can be used for DNA sequencing as long as it contains the human genome. · Base pairs: Adenine & Thymine; Guanine & Cytosine. Adenine is complimentary to thymine and guanine is complimentary to cytosine. . The structure of DNA is stable because of the sugar phosphate backbone. . The hydrogen bonds attach the two strands in DNA by holding the base pairs together, causing DNA to have a double-helix structure. Sequencing by Synthesis(Step by Step): 1. The two strands of DNA are separated into single-stranded templates through library construction. 2. Each of these DNA templates get deposited on a slide. 3. DNA polymerase is used to synthesize molecules. It is added onto the sequencing machine along with DNA bases. These DNA bases have speed bumps(terminators), meaning nothing can be attached on top of them(including other nucleotides to bind with the little DNA strands) 4. The DNA bases compete to attach to a complimentary base. The DNA polymerase binds nucleotides to their complementary bases. 5. An area camera is used to take a snapshot of the top of the templates. 6. Remove terminators and then repeat steps 3-5(one cycle of sequencing) The length of the number of nucleotides = number of DNA sequencing cycles(ex. 500 nucleotides = 500 DNA sequencing cycles). These DNA sequencing cycles are directly related to sequencing read length. One base is sequenced per cycle, the total number of cycles indicates the maximum number of bases that can be sequenced.
Sequencing by synthesis(more details): · Billions of templates on a slide · For massively parallel sequencing, the photograph captures all templates simultaneously. The high resolution camera picks up these colors. · The critical difference between Sanger sequencing and NGS is sequencing volume. While the Sanger method only sequences a single DNA fragment at a time, NGS is massively parallel, sequencing millions of fragments simultaneously per run. · Terminators are "speed bumps", keeping reactions in sync.