Module 2.1 Strategies for Sequencing Genomes.pdf

Module 2.1 Strategies for Sequencing Genomes Learning Outcomes After completing this module you will be able to: Describe what a genome is Explain the strategy used to decode a genome Explain why genomic maps and map features are a necessary aid in genome sequencing Describe...

Module 2.1 Strategies for Sequencing Genomes Learning Outcomes After completing this module you will be able to: Describe what a genome is Explain the strategy used to decode a genome Explain why genomic maps and map features are a necessary aid in genome sequencing Describe what a genomic library is and how it is used in genomic sequencing Define clone-contig and shotgun sequencing approaches  What is a Genome Nucleic acid sequence, (1) encodes information for production of an organism, and (2) the genetic material transferred to new generations A genome contains functional units called genes, arranged linearly on chromosomes Genome sequences are generally stable, but changes arise from errors of replication and mutation, which is one of two forces driving evolution (the other being natural selection)  Analyzing Genomes In the Age of Genomics Advances in sequencing technology have resulted in a surge of decoded whole genome sequences http://www.ncbi.nlm.nih.gov/ Genomes are huge (human 3.2 billion bases) State-of-the-art sequencing produces at most 750 b of information in a single run So, do the math 3,200,000,000 ÷ 750 b = 4,300,000 sequencing runs Image Stitching  Google Maps …is a massive image stitch created by superimposing enormous numbers of individual remote sensing images. Image stitching is verified by ground truthing. That’s how street-views are produced. Analogous to markers on a genomic map.  Contiguous sequence- Contig Figure 3.1 Genomes 3 (© Garland Science 2007)  In this example, the fragment overlap is 6 b. A 6 b sequence can have any of the 4 nitrogenous bases at each position. Therefore, the frequency would be 46 = 4096 b. One would expect this 6 b sequence to exist 781,250 times in the human genome… 3.2 billion ÷ 4096. Put another way, you would need between a 15 to 16 b overlap to ensure a single occurrence in the human genome. Genomes are Assembled from Sequencing of Genomic Libraries  1. Cloning into bacteria or yeast DNA Fragments are Replicated in the Microtiter Wells Using Two Different Methods. 2. Cell-free cloning using Polymerase Chain Reaction. (to be covered later).  Fragment ends can be sequenced Genome Assemblage Relies on Entire clones can be Varied sequenced, better if you know where the Strategies of clones come from Sequencing and Contig Assembly Entire libraries can be sequenced Two Methods Have Been Used Clone Contig Method Is older, uses a traditional step-wise approach of sequencing clones that have been placed on a genomic map Shotgun Sequencing More recent, uses computer assistance to find overlaps in large amounts of randomly generated sequence to produce contigs, that are then placed on a genomic map Figure 3.3 Genomes 3 (© Garland Science 2007) The biggest problem with a pure shotgun approach is: TANDEMLY REPEATED GENOME-WIDE SEQUENCES REPEATED SEQUENCES Figure 3.2a Genomes 3 (© Garland Science 2007) Figure 3.2b Genomes 3 (© Garland Science 2007)  How can the problem of tandem and genome-wide repeats be solved when sequencing genomes? What are the pros and cons of Recitation clone-contig and shotgun sequencing approaches? Discussion Why are genomic map features Problems important for both clone-contig and shotgun sequencing approaches? What kinds of map features would be useful for genome sequencing?