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3-7-4. Whole-Genome SequencingLearning Objectives
Although there have been significant advances in the medical sciences in recent years, doctors are still confounded by some diseases, and they are using whole-genome sequencing to get to the bottom of the problem. For example, whole-exome sequencing is a lower-cost alternative to whole genome sequencing. In exome sequencing, only the coding, exon-producing regions of the DNA are sequenced. In 2010, whole-exome sequencing was used to save a young boy whose intestines had multiple mysterious abscesses. The child had several colon operations with no relief. Finally, whole-exome sequencing was performed, which revealed a defect in a pathway that controls apoptosis (programmed cell death). A bone-marrow transplant was used to overcome this genetic disorder, leading to a cure for the boy. He was the first person to be successfully treated based on a diagnosis made by whole-exome sequencing. Today, human genome sequencing is more readily available and can be completed in a day or two for about $1000. Strategies Used in Sequencing ProjectsThe basic sequencing technique used in all modern day sequencing projects is the chain termination method (also known as the dideoxy method), which was developed by Fred Sanger in the 1970s. The chain termination method involves DNA replication of a single-stranded template with the use of a primer and a regular ![]() Figure 1: A dideoxynucleotide is similar in structure to a deoxynucleotide, but is missing the 3' hydroxyl group (indicated by the box). When a dideoxynucleotide is incorporated into a DNA strand, DNA synthesis stops. ![]() Figure 2: Frederick Sanger's dideoxy chain termination method is illustrated. Using dideoxynucleotides, the DNA fragment can be terminated at different points. The DNA is separated on the basis of size, and these bands, based on the size of the fragments, can be read. Early Strategies: Shotgun Sequencing and Pair-Wise End SequencingIn Originally, shotgun sequencing only analyzed one end of each fragment for overlaps. This was sufficient for sequencing small genomes. However, the desire to sequence larger genomes, such as that of a human, led to the development of double-barrel shotgun sequencing, more formally known as Next-generation SequencingSince 2005, automated sequencing techniques used by laboratories are under the umbrella of Evolution ConnectionComparing SequencesA sequence alignment is an arrangement of proteins, DNA, or RNA; it is used to identify regions of similarity between cell types or species, which may indicate conservation of function or structures. Sequence alignments may be used to construct phylogenetic trees. The following website uses a software program called BLAST (basic local alignment search tool). Under “Basic Blast,” click “Nucleotide Blast.” Input the following sequence into the large "query sequence" box: ATTGCTTCGATTGCA. Below the box, locate the "Species" field and type "human" or "Homo sapiens". Then click “BLAST” to compare the inputted sequence against known sequences of the human genome. The result is that this sequence occurs in over a hundred places in the human genome. Scroll down below the graphic with the horizontal bars and you will see short description of each of the matching hits. Pick one of the hits near the top of the list and click on "Graphics". This will bring you to a page that shows where the sequence is found within the entire human genome. You can move the slider that looks like a green flag back and forth to view the sequences immediately around the selected gene. You can then return to your selected sequence by clicking the "ATG" button. Use of Whole-Genome Sequences of Model OrganismsThe first genome to be completely sequenced was of a bacterial virus, the bacteriophage fx174 (5368 base pairs); this was accomplished by Fred Sanger using shotgun sequencing. Several other organelle and viral genomes were later sequenced. The first organism whose genome was sequenced was the bacterium Haemophilus influenzae; this was accomplished by Craig Venter in the 1980s. Approximately 74 different laboratories collaborated on the sequencing of the genome of the yeast Saccharomyces cerevisiae, which began in 1989 and was completed in 1996, because it was 60 times bigger than any other genome that had been sequenced. By 1997, the genome sequences of two important model organisms were available: the bacterium Escherichia coli K12 and the yeast Saccharomyces cerevisiae. Genomes of other model organisms, such as the mouse Mus musculus, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis. elegans, and humans Homo sapiens are now known. A lot of basic research is performed in model organisms because the information can be applied to genetically similar organisms. A
Link to Learning
Click through each step of genome sequencing at this site. Uses of Genome Sequences
In addition to disease and medicine, genomics can contribute to the development of novel enzymes that convert biomass to biofuel, which results in higher crop and fuel production, and lower cost to the consumer. This knowledge should allow better methods of control over the microbes that are used in the production of biofuels. Genomics could also improve the methods used to monitor the impact of pollutants on ecosystems and help clean up environmental contaminants. Genomics has allowed for the development of agrochemicals and pharmaceuticals that could benefit medical science and agriculture. It sounds great to have all the knowledge we can get from whole-genome sequencing; however, humans have a responsibility to use this knowledge wisely. Otherwise, it could be easy to misuse the power of such knowledge, leading to discrimination based on a person's genetics, human genetic engineering, and other ethical concerns. This information could also lead to legal issues regarding health and privacy. Section SummaryWhole-genome sequencing is the latest available resource to treat genetic diseases. Some doctors are using whole-genome sequencing to save lives. Genomics has many industrial applications including biofuel development, agriculture, pharmaceuticals, and pollution control. The basic principle of all modern-day sequencing strategies involves the chain termination method of sequencing. Although the human genome sequences provide key insights to medical professionals, researchers use whole-genome sequences of model organisms to better understand the genome of the species. Automation and the decreased cost of whole-genome sequencing may lead to personalized medicine in the future. Review QuestionsExercise 1The chain termination method of sequencing:
Show/Hide Solution A Exercise 2Whole-genome sequencing can be used for advances in:
Show/Hide Solution D Exercise 3Sequencing an individual person’s genome
Show/Hide Solution D Exercise 4What is the most challenging issue facing genome sequencing?
Show/Hide Solution B Glossarychain termination method contig deoxynucleotide dideoxynucleotide DNA microarray genome annotation model organism next-generation sequencing shotgun sequencing whole-genome sequencing
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