Agenda • Background • Next Generation Sequencing • Sequence Assembly • Sequence Alignment • Traditional Alignment Algorithms • Next Generation Alignment Algorithms • Conclusion Andy Nagar 2 Background • Earlier sequencing methods were based on Sanger sequencing, which goes back to the 1970s. • Sequencing was slow, bases were read one at a time. • Separation is done by electrophoresis. • Readout by fluorescent tags ...
DNA fragment sequencing In order to establish exactly what changes have occurred in the gene under study, it will be necessary determine the DNA sequence of that gene. This is now a routine procedure in molecular biology laboratories, extending not just to individual genes or fragments but to the complete sequence of the DNA (the genome) of the organism. The two main in vitro DNA ...
Work flow of conventional versus second- generation sequencing (a) With high-throughput shotgun Sanger sequencing, genomic DNA is fragmented, then cloned to a plasmid vector and used to transform E. coli. For each sequencing reaction, a single bacterial colony is picked and plasmid DNA isolated. Each cycle sequencing reaction takes place within a microliter-scale volume, generating a ladder of ddNTP-terminated, dye-labeled products, which are subjected to ...
Our Mtb DNA on the Tapestation MinION fastq file 2 records – 576 bp; 1316 bp Illumina fastq file all records ~100 bp There are 3 main approaches to sequencing Market Approach / USP Read Achievements Ideal for: dominance length st 1 Generation Applied Sequencing by 1 kb Most primary PCR products (Sanger) Biosystems synthesis (SBS) drafts, e.g. (genotyping) - Low throughput Mtb, human ...
Courtesy of CRC Press/Taylor & Francis Group Figure 11.2 Sanger sequencing with fluorescent nucleotides. Rather than using four separate radioactive nucleotides in the sequencing reactions, use of nucleotides each labeled with a different type of fluorescence reduced both the number of reactions (top) and the number of lanes on the polyacrylamide gel (middle). Fluorescence also enabled automated detection (bottom), reducing human error in ...
Sanger (dideoxy) DNA sequencing procedure overview •The reaction is a DNA synthesis reaction which involves: •a high concentration of regular deoxynucleotides •a low concentration of dye-labeled dideoxynucleotides •Each of the four dideoxynucleotides (A,T,C,G) is labeled with a different color dye. •After the DNA synthesis reaction, the DNA produced is imaged during electrophoresis to detect dye colors associated ...
DNA sequencing How we obtain the sequence of nucleotides of a species ACGTGACTGAGGACCGTG CGACTGAGACTGACTGGGT CTAGCTAGACTACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT Which representative of the species? Which human? Answer one: Answer two: it doesn’t matter Polymorphism rate: number of letter changes between two different members of a species Humans: ~1/1,000 Other organisms have much higher polymorphism rates Population size! Ancient sequencing technology – Sanger ...
DNA sequencing ACGTGACTGAGGACCGTG CGACTGAGACTGACTGGGT CTAGCTAGACTACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT DNA sequencing Determination of nucleotide sequence Two similar methods: 1. Maxam and Gilbert method 2. Sanger method They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in length by a single nucleotide at the other end This mixture of oligonucleotides is separated by ...
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