Lecture 14: DNA Sequencing - Rhodes College
Lecture 14:DNA SequencingStudy Chapter 8.910/18/2013COMP 465 Fall 20131
DNA Sequencing Shear DNA intomillions of smallfragments Read 500 – 700nucleotides at a timefrom the smallfragments(Sanger method)10/18/2013COMP 465 Fall 20132
Fragment Assembly Assembles the individual overlapping shortfragments (reads) into a genomic sequence Shortest Superstring problem from last time isan overly simplified abstraction Problems:– DNA read error rate of 1% to 3%– Can’t separate coding and template strands– DNA is full of repeats Let’s take a closer look10/18/2013COMP 465 Fall 20133
Construction of Repeat Graph Construction of repeat graph from k – mers:emulates an SBH experiment with a huge(virtual) DNA chip. Breaking reads into k – mers: Transformsequencing data into virtual DNA chip data.10/18/2013COMP 465 Fall 20134
Construction of Repeat Graph (cont’d) Error correction in reads: “consensus first”approach to fragment assembly. Makes reads(almost) error-free BEFORE the assembly evenstarts. Using reads and mate-pairs to simplify therepeat graph (Eulerian Superpath Problem).10/18/2013COMP 465 Fall 20135
Approaches to Fragment AssemblyFind a path visiting every VERTEX exactlyonce in the OVERLAP graph:Hamiltonian path problemNP-complete: algorithms unknown10/18/2013COMP 465 Fall 20136
Approaches to Fragment Assembly(cont’d)Find a path visiting every EDGE exactly oncein the REPEAT graph:Eulerian path problemLinear time algorithms are known10/18/2013COMP 465 Fall 20137
Making Repeat Graph Without DNA Problem: Construct the repeat graph from acollection of reads.? Solution: Break the reads into smaller pieces.10/18/2013COMP 465 Fall 20138
Repeat Sequences: Emulating aDNA Chip Virtual DNA chip allows the biological problemto be solved within the technological constraints.10/18/2013COMP 465 Fall 20139
Repeat Sequences: Emulating aDNA Chip (cont’d) Reads are constructed from an original sequencein lengths that allow biologists a high level ofcertainty. They are then broken again to allow thetechnology to sequence each within a reasonablearray.10/18/2013COMP 465 Fall 201310
Minimizing Errors If an error exists in one of the 20-mer reads, theerror will be perpetuated among all of thesmaller pieces broken from that read.10/18/2013COMP 465 Fall 201311
Minimizing Errors (cont’d) However, that error will not be present in theother instances of the 20-mer read. So it is possible to eliminate most point mutationerrors before reconstructing the originalsequence.10/18/2013COMP 465 Fall 201312
Conclusion from Previous Lecture Graph theory is a vital tool for solving biologicalproblems Wide range of applications, includingsequencing, motif finding, protein networks, andmany more10/18/2013COMP 465 Fall 201313
DNA Sequencing Timeline10/21/2013COMP 465 Fall 201314
Generations of Sequences10/22/2013COMP 465 Fall 201315
High-Throughput Sequencing Also referred to as Next-Generation Sequencing Parallelize the sequencing process, producingthousands or millions of sequences concurrently Lower the cost of DNA sequencing beyond whatis possible with standard dye-terminatormethods. In ultra-high-throughput sequencing as many as500,000 sequencing-by-synthesis operations maybe run in parallel10/21/2013COMP 465 Fall 201316
10/21/2013COMP 465 Fall 201317
Next Generation Sequencing:Amplified Single Molecule Sequencing10/22/2013COMP 465 Fall 201318
Next Generation Sequencing:Amplified Single Molecule Sequencing10/22/2013COMP 465 Fall 201319
454 Sequencing10/22/2013COMP 465 Fall 201320
454 Sequencing10/22/2013COMP 465 Fall 201321
454 Sequencing / Pyrosequencing10/22/2013COMP 465 Fall 201322
454 Sequencing / Pyrosequencing10/22/2013COMP 465 Fall 201323
454 Sequencing / Pyrosequencing10/22/2013COMP 465 Fall 201324
SOLiD10/22/2013COMP 465 Fall 201325
SOLiD10/22/2013COMP 465 Fall 201326
Sequencing By Ligation10/22/2013COMP 465 Fall 201327
Sequencing By Ligation10/22/2013COMP 465 Fall 201328
Sequencing By Ligation10/22/2013COMP 465 Fall 201329
Sequencing By Ligation10/22/2013COMP 465 Fall 201330
Sequencing By Ligation10/22/2013COMP 465 Fall 201331
Sequencing By Ligation10/22/2013COMP 465 Fall 201332
Sequencing By Ligation10/22/2013COMP 465 Fall 201333
Sequencing By Ligation10/22/2013COMP 465 Fall 201334
Sequencing By Ligation10/22/2013COMP 465 Fall 201335
Illumina10/22/2013COMP 465 Fall 201336
Illumina10/22/2013COMP 465 Fall 201337
Illumina10/22/2013COMP 465 Fall 201338
Which Next-Gen Sequencer toChoose for your Project?10/22/2013COMP 465 Fall 201339
Mouse Genomes Project mes/lookseq/index.pl?show 8:101738730101738871,paired pileup&lane C3H HeJ.bam&width 900&win 141&display perfect single inversions pairlinks potsnps uniqueness gc coverage orientation annotation gc coverage &maxdist 100010/22/2013COMP 465 Fall 201340
Sequence Comparisons10/22/2013COMP 465 Fall 201341
Human Genome Project In Dec. 1, 1999, researchers in the Human GenomeProject announced the complete sequencing of the DNAmaking up human chromosome 22. In 2000, the completion of a “working draft” DNAsequence of the human genome was announced. Special issues of Nature and Science came out inFebruary of 2001 with the complete working drafthuman genome.10/22/2013COMP 465 Fall 201342
Human Genome Project International HapMap Project began in 2002. Special issue of Nature Human GenomeCollection (2006) On June 13, 2013, The U.S. Supreme Courtruled that naturally occurring DNA cannot bepatented, but that synthetically created cDNAis patent-eligible.10/22/2013COMP 465 Fall 201343
References Simons, Robert W. Advanced Molecular Genetics Course, UCLA(2002). Batzoglou, S. Computational Genomics Course, Stanford University(2006). http://ai.stanford.edu/ serafim/CS262 2006/ Vierstraete, Andy. Next Generation Sequencing, University of Ghent.http://users.ugent.be/ avierstr/nextgen/nextgen.html10/22/2013COMP 465 Fall 201344
Next Time Protein Sequencing Sections 8.10-8.1510/21/2013COMP 465 Fall 201345
Also referred to as Next-Generation Sequencing Parallelize the sequencing process, producing thousands or millions of sequences concurrently Lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods. In ultra-high-throughput sequencing as many as 500,000 sequencing-by-synthesis operations may
Introduction of Chemical Reaction Engineering Introduction about Chemical Engineering 0:31:15 0:31:09. Lecture 14 Lecture 15 Lecture 16 Lecture 17 Lecture 18 Lecture 19 Lecture 20 Lecture 21 Lecture 22 Lecture 23 Lecture 24 Lecture 25 Lecture 26 Lecture 27 Lecture 28 Lecture
DNA Sequencing Troubleshooting Guide. There are a number of factors that can lead to less than perfect DNA sequencing results. In this guide, we explain some of the common problems encountered, and outline ways in which these problems can be overcome. Below is an example of a normal sequencing result. Shown here is the raw data signal
NA sequencing has two intertwined histories—that of the under - lying technologies and that of the breadth of problems for which it has proven useful. Here we first review major developments in the history of DNA sequencing technologies (Fig. 1). Next we consider the trajectory of DNA sequencing applications (Fig. 2). Finally, we discuss
Genetic transformation and DNA DNA is the genetic material in bacterial viruses (phage) The base-pairing rule DNA structure. 2. Basis for polarity of SS DNA and anti-parallel complementary strands of DNA 3. DNA replication models 4. Mechanism of DNA replication: steps and molecular machinery
Recombinant DNA Technology 3. Recombinant DNA Technology 600 DNA ISOLATION AND PURIFICATION Basic to all biotechnology research is the ability to manipulate DNA. First and foremost for recombinant DNA work, researchers need a method to isolate DNA from different organisms. Isolating DNA from bacteria is the easiest procedure because bacterial cells
The tools of biotechnology include DNA sequencing, using recombinant DNA, DNA synthesis and genome editing. However, editing genes could have unintended ecological impacts, be used maliciously or mutate unexpectedly. DNA s e q u e n c i n g In DNA sequencing, we find out the specific code of instructions or genes a cell, plant or person may have.
Chapter 20: Biotechnology: The DNA Toolbox Sequencing of the human genome was completed by 2007 DNA sequencing has depended on advances in technology, starting with making recombinant DNA – In recombinant DNA, nucleotide sequences from two different sources, often two
avanzados de Alfredo López Austin, Leonardo López Lujan, Guilhem Olivier, Carlos Felipe Barrera y Elsa Argelia Guerrero con la intención de mostrar si existió ó no el sacrificio humano entre los aztecas y si los hubo con qué frecuencia y crueldad. Por otra parte, he de mencionar que la elaboración de este trabajo ha sido una ardua tarea de síntesis de diferentes fuentes sobre la .
