CS 262:2009 Lecture 9: DNA Sequencing / Scribe: Robert BruggnerBackgroundDeoxyribonucleic acid (DNA) is a self-replicating molecule present in nearly all living organisms that contains all of our genetic information. Each molecule of DNA contains complementary strings of nucleotides that encode all the necessary information for an organism to develop, live, copy itself, and pass along the same genetic information to its offspring. As this is quite a complex process, a large amount of DNA is necessary to provide all the required instructions. Modern humans have approximately 3 billion nucleotides in their DNA sequence contained in 23 smaller divisions of sequence called chromosomes. The entirety of this sequence is referred to as an organismʼs genomic sequence and is found in nearly all of an organismʼs cells. Because an organismʼs DNA contains the foundational information necessary to produce, regulate, and control smaller components of life within that organism, weʼre naturally interested in learning the exact sequence of that organismʼs DNA is and what each of the smaller functional pieces do. DNA Sequencing is a process that allows us to determine the order of nucleotides that makes up a molecule of DNA. Learning the sequence of an organismʼs DNA enables analysis of that organismʼs genetic code using cheap, powerful computational methods in lieu of expensive and complex laboratory procedures. In an ideal scenario, one would simply be able to take any bit of biological material that contains DNA, put it into a machine, and the machine would print out a long sequence of letters representing the exact sequence of DNA for that organism. Unfortunately, the process is quite complex and difficulties are discussed later in this document. White Blood Cells"" " DNA Sequencer" " DNA Sequence"AGCTACGT...Which individual do you sequence?Although nearly identical, the DNA among individuals of a species tends to vary slightly due to evolutionary and environmental pressures as well as random mutations. Accordingly, when embarking on a genome sequencing project, one must choose one or more individuals whoʼs sequenced DNA will be representative of the species. The question then arrises of which individual(s) to sequence. During the 1990ʼs, there were two independently directed programs attempting to sequence the human genome. The first, the government-sponsored Human Genome Project, used DNA collected from 12 anonymous regional individuals as its source for sequencing. The second, led by Dr. Craig Venter of Celera Genomics, attempted to sequence the DNA of a single individual - Dr. Venter himself. So ultimately, which genomic sequence was more representative of the current human population? As it turns out, it doesnʼt actually matter which individuals we choose to sequence because any two individual human genomes are already over 99.5% similar. Another way of looking at this is that, on average, humans DNA only differs by a single nucleotide out of thousand. This rate of differentiation between to individuals of a species is known as the Polymorphism Rate or in other words, how often we expect to find differentiated DNA sequence between two individuals. A polymorphism can either be a single base pair that differs between individuals ( Single Nucleotide Polymorphism or SNP) or a repetitive stretch of DNA that varies in repeat copy number between individuals (copy number variation). The latter accounts for more variation between individuals but itʼs also less clear how those variations manifest themselves in the organismʼs phenotype. The heterozygosity of a population (the proportion of differences across two individuals) can be calculated by: " h = 4*N*µ/(1+4*N*µ) where:" N = The total population size and" µ = Species mutation rate (proportion of bp that differ between parent and child)Given the known amount of similarity among human genomes, one can simply sequence an individual (or set of individuals), and treat the found DNA sequence as the Reference Genome and describe all other individuals in terms of their variance from the reference. In terms of polymorphism rate, the human species has a high rate of conservation between individuals. DNA of other organisms, such as Ciona savignyi, differ by as many as one base pair out of ten between individuals. Such variation is attributed to the organismʼs massive population size. What accounts for such a descrepancy in polymorphism rates between C. savignyi and H. sapiens?Human Population MigrationsThe current most widely accepted theory of human evolution, the Out of Africa Replacement Theory postulates that a population of humans that originated in Africa, slowly expanded and replaced other human populations (such as Neandertal) approximately 40,000 years ago. Thus, since contemporary human individuals evolved from such a small, interbred population, genetic variance among modern individuals has been greatly limited. If the initial diversity of individuals within the human populations of Africa looked similar to this...... then itʼs possible to imagine that only a fraction of that diverse population would migrate away from Africa during the replacement period. Accordingly, the oldest populations of Homo sapiens in Africa have the highest polymorphism rate between individuals while relatively new populations have less. Theory Support The Out of Africa Replacement Theory is supported partially through analysis of divergence in DNA between parents and children. All male individuals inherit a “Y” chromosome from their father and given that we know (approximately) the rate of DNA mutation between two generations, we can calculate the the number of generations that have occurred since any two pair of y-chromosomes were last identical. Effectively, this point of coalescence will tell us the most recent common ancestral father between any two individuals. Likewise, we can examine mitochondrial DNA, DNA inherited only fromoneʼs mother, in the same manner to determine the most recent common ancestral mother between any two individuals. From such analysis, itʼs currently theorized that the most common mother among all current human individuals most likely occurred approximately 150,000 years ago and the most recent father approximately 70,000 years ago. The difference in the time between a common mother and a common father has is often attributed to the fact that historically, males have tended to 1) father more more kids
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