Introduction to Bioinformatics. Lecture 20: Sequencing genomes

Transcription

1 Introduction to Bioinformatics Lecture 20: Sequencing genomes

2 Nucleic Acid Basics Nucleic Acids Are Polymers Each Monomer Consists of Three Moieties: Nucleotide A Base + A Ribose Sugar + A Phosphate Nucleoside A Base Can be One of the Five Rings:

3 Pyrimidines Purines Pyrimidines and Purines can Base-Pair (Watson-Crick Pairs)

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5 Unlike three dimensional structures of proteins, DNA molecules assume simple double helical structures independent of their sequences. There are three kinds of double helices that have been observed in DNA: type A, type B, and type Z, which differ in their geometries. The double helical structure is essential to the coding function of DNA. Watson (biologist) and Crick (physicist) first discovered the double helix structure in 1953 by X-ray crystallography. RNA, on the other hand, can have as diverse structures as proteins, as well as simple double helix of type A. The ability of being both informational and diverse in structure suggests that RNA was the prebiotic molecule that could function in both replication and catalysis (The RNA World Hypothesis). In fact, some viruses encode their genetic materials by RNA (retrovirus)

6 Forces That Stabilize Nucleic Acid Double Helix There are two major forces that contribute to stability of helix formation Hydrogen bonding in base-pairing Hydrophobic interactions in base stacking 5 3 Same strand stacking cross-strand stacking 3 5

7 Types of DNA Double Helix Type A: major conformation of RNA, minor conformation of DNA; Type B: major conformation of DNA; Type Z: minor conformation of DNA A Narrow tight 5 3 B Wide Less tight 5 3 Z Left-handed Least tight 5

8 Three Dimensional Structures of Double Helices A-DNA A-DNA Minor Groove Major Groove A-RNA

9 Secondary Structures of Nucleic Acids DNA is primarily in duplex form. RNA is normally single stranded which can have a diverse form of secondary structures other than duplex.

10 More Secondary Structures of Nucleic Acids Pseudoknots: Source: Cornelis W. A. Pleij in Gesteland, R. F. and Atkins, J. F. (1993) THE RNA WORLD. Cold Spring Harbor Laboratory Press.

11 3D Structures of RNA: Transfer RNA Structures Secondary Structure of trna Tertiary Structure of trna TψC Loop Variable loop D Loop Anticodon Stem Anticodon Loop

12 3D Structures of RNA: Ribosomal RNA Structures Secondary Structure Of large ribosomal RNA Tertiary Structure Of large ribosome subunit Ban et al., Science 289 ( ), 2000 rrna Secondary Structure Based on Phylogenetic Data

13 DNA Sequencing Chain Termination Method Sanger, 1977 single stranded DNA, ~800b Method: Electrophoresis can separate DNA molecules differing 1bp in length Dideoxynucleotide (ddntp) are used -which stop replication

14 ddnucleotides dda, ddt, ddc, ddg Each type marked with fluorescent dye When incorporated into DNA chain stops replication

15 Chain Termination Method, An Outline Replication Obtaining ssdna Add a (universal) primer Start replication in a soup of A,T,C,G Continously add tiny amounts of dda, ddt, ddc, ddg gradually stopping all the processes

16 Chain Termination Method, Reading the Sequence Running through electrophoresis gel Four types of ddntp have four different fluorescent labels Automated reading See:

17 Chain Termination Method, Results Signal Electrophoresis and laser beam scanning time Electropherogram fragment size

18 Shotgun Method - Overview Cut genome into short fragments Sequence DNA fragments Create contigs Contig - continous set of overlapping sequences Gap!

19 Shotgun Method The shotgun approach to sequence assembly. The DNA molecule is broken into small fragments, each of which is sequenced. The master sequence is assembled by searching for overlaps between the sequences of individual fragments. In practice, an overlap of several tens of base pairs would be needed to establish that two sequences should be linked together.

20 Shotgun Method Contig Construction Two DNA sequences: X=CTATCA Y=AGTAT How do they overlap? X Y Try to apply dynamic programming or X Y

21 Shotgun Method Contig Construction by Dynamic Programming 2 1

22 Shotgun Method Haemophilus Influenzae Sequencing Extract DNA DNA library Electrophoresis Sonicate 1.5-2kb Sequence Construct contigs Sequenced

23 Shotgun Method - Filling in gaps Contig Gap Contig Gap Contig Probe libraries Scaffold A series of sequence contigs separated by sequence gaps.

24 Shotgun Method - Pros and Cons Pros Human labour reduced to minimum Cons Computationally demanding O(n 2 ) comparisons High error rate in contig construction Repeats as the main problem

25 Shotgun Method Repeats as the main problem

26 Shotgun vs. Hierarchical Method Celera vs. Human Genome Project Hierarchical (top-down) assembly: The genome is carefully mapped Shotgun into large chunks of 150kb Exact location of each chunk is known Each piece is again shotgunned into 2kb and sequenced

27 Shotgun vs. Hierarchical Method Shotgun bottom-up Hierarchical top-down

28 New Sequencing Methods Sequencing By Hybridization Check which from all possible fragments of length k (k-tuples) hybridize to the sequence TAA AAG AGC ATTCG TAAGC

29 Wrapping up Nucleotide, DNA, RNA basics (sequence, structure) DNA Sequencing Sanger method Shotgun sequencing Hierarchical assembly Contigs, scaffolds, Dynamic Programming