3/17/2003 bioinformatics: merging biological and computational skills eric rouchka, d.sc. university...

3/17/2003 Bioinformatics: Merging Biological and Computational Skills Eric Rouchka, D.Sc. University of Louisville

Overview of Genome Sequencing Progress

Eric C. Rouchka, D.Sc.

Bioinformatics Journal Club

October 1, 2003


DNA Sequences

• DNA: double stranded helix

• Composed of 4 bases: A,C,G,T

• Genome: linear chain of bases– Humans: 22 Autosome pairs, 2

sex chromosomes, 3.2 billion bases

(Image source: http://www.ebi.ac.uk/)


Double Helix

• Two complementary DNA strands form a stable DNA double helix

• A, T are complements; G, C are complements

Image source; www.ebi.ac.uk/microarray/ biology_intro.htm


Brief History of Sequencing

• Discovery of Complementary Bases– Erwin Chargaff, 1950

• Discovery of DNA Double Helix– 1953 – only 50 years ago

– James Watson– Francis Crick– Rosland Franklin

Image: www.simr.org.uk/pages/biotechnology/ biotechnology_2.html


DNA

RNA

PROTEIN

Central Dogma


RNA• Ribonucleic Acid

• Similar to DNA

• Thymine (T) is replaced by uracil (U)

• RNA can be:– Single stranded– Double stranded– Hybridized with DNA


RNA

• RNA is generally single stranded

• Forms secondary or tertiary structures

• Important in a variety of ways, including protein synthesis


How Central Dogma Works

• DNA “transcribed” into SS mRNA

• mRNA “translated” into protein using tRNA– Triplet bases (codons) used to code amino

acids– 3 mRNA bases code one amino acid


History Of Genetic Code

• Genetic Code Completely uncovered (1965)– Marshall Nierenberg


Genetic Code

• 4 possible bases (A, C, G, U)• 4 * 4 * 4 = 64 possible codon sequences• Start codon: AUG• Stop codons: UAA, UAG, UGA• 61 codons to code for amino acids (AUG as

well)• 20 amino acids – redundancy in genetic code


20 Amino Acids• Glycine (G, GLY)• Alanine (A, ALA)• Valine (V, VAL)• Leucine (L, LEU)• Isoleucine (I, ILE)• Phenylalanine (F, PHE)• Proline (P, PRO)• Serine (S, SER)• Threonine (T, THR)• Cysteine (C, CYS)• Methionine (M, MET)• Tryptophan (W, TRP)• Tyrosine (T, TYR)• Asparagine (N, ASN)• Glutamine (Q, GLN)• Aspartic acid (D, ASP)• Glutamic Acid (E, GLU)• Lysine (K, LYS)• Arginine (R, ARG)• Histidine (H, HIS)• START: AUG• STOP: UAA, UAG, UGA


tRNA structure

http://www.tulane.edu/~biochem/nolan/lectures/rna/frames/trnabtx2.htm


Protein Structure

Image source; www.ebi.ac.uk/microarray/ biology_intro.htm


Brief History of Sequencing

• First Protein Sequence– ~1955 Bovine Insulin (Fred Sanger)

• First DNA Sequence– ~1965 yeast alanine tRNA (77 bases)

• Development of DNA sequencing – Maxam-Gilbert and Sanger Methods (1977)


Sanger Sequencing Method

• (Quicktime Movie)

• SOURCE: Molecular Cell Biology


Improving Sanger’s Method

• Dideoxynucleosides fluorescently labeled (1986)– Reaction cut by ¼

• Sequencing Automated by machine (1986)

• Laser detects fluorescence


Image Source: plantbio.berkeley.edu/ ~bruns/tour3.html


Genetic Mapping

• Sex-linked genes studied since early 1900s

• Gene mapping takes off in late 1970s– David Botstein (RFLPs 1978)

• 1979: 579 Genes Mapped• 2003 ~30,000 Genes Mapped

– Mapping of Huntington’s Disease (First Diseased Gene)• Triplet Repeat• 1983• Nancy Wexler


Mapping of Markers

• Sequence Tagged Sites (STS)– Sequences occurring only once in the

human genome

– Help to map locations

– 52,000 STS in Humans• ~ 1 every 62,000 bases


Cloning Techniques

• Plasmid Cloning Introduced (1973)

– Region of Interest duplicated by inclusion

• YAC Chromosomes described (1987)

• BACs introduced (1992)

• 30,000 to 100,000 bases can be cloned

pACYC1773941 bp

KN(R)

AP r

P15A ORI

TN3 INV RPT

TN903 INV RPT

TN903 INV RPT

TN3 REPR FRAG

Apa LI (3815)

Bam HI (3321)

Cla I (2046)

Hin dIII (2473)

Pst I (304)

Sma I (2229)

Xma I (2227) Ava I (1953)

Ava I (2227)


Hierarchical (Clone-based) Approach

• Know location of 30,000 – 100,000 bp region• Break into 500-700 bp fragments• Sequence Fragments• Assemble based on similarity• ~8-10x coverage

• Current Price: $0.09 / base


Hierarchical (clone-based) approach

• generate overlapping set of clones• select a minimum tiling path• shotgun sequence each clone


Hierarchical (clone-based) approach

• MINUS– map generation requires resources, time

and money– Some regions not cloned

• PLUS– easier to assemble smaller pieces– less chance for assembly error


Shotgun Sequencing Approach

• Developed 1991 TIGR– Craig Venter, Hamilton Smith

• Break genome into millions of pieces– Sequence each piece– Reassemble into full genomes


Whole Genome Shotgun Approach

• reads generated directly from a whole-genome library

• assemble the genome all at once


Whole Genome Shotgun Approach

• MINUS– more prone to assembly error– computationally intensive– cannot effectively handle repeats

• PLUS– Less overhead time up front


Base calling and Assembly Software

• PHRED and PHRAP Developed (1988)– PHRED: Base calling software– PHRAP: Assists in assembly of sequenced

data


Available Assemblers

• SEQAID (Peltola et al., 1984)• CAP (Huang, 1992)• PHRAP (Green, 1994)• TIGR Assembler (Sutton et al., 1995)• AMASS (Kim et al., 1999)• CAP3 (Huang and Madan, 1999)• Celera Assembler (Myers et al., 2000)• EULER (Pevzner et al., 2001)• ARACHNE (Batzoglou et al., 2002)


History of Genome Projects

• First Genome Sequence

– FX174 Phage 5,386 bp; 9 proteins (1980)

• Haemophilus Influenzea Sequenced

– First non-viral genome (1.8 MB) (1995)



• Saccharomyces cereviseae sequenced

– First eukaryotic genome (12.1 MB) (1996)

• Caenorhabditis elegans sequence released

– First animal genome 200 MB (1998)



• Arabidopsis thaliana sequence released

– First publicly available plant genome (1999)

• Rough Draft of Human Genome Reported (2001)– “Finished” 2003


Human Genome Project

• Began in 1990 (US DOE – 15 years)– Identify all genes in human DNA– Determine sequence of human genome– Develop faster sequencing technologies– Develop tools for data analysis– ELSI


Microbial Genomes

• 122 Complete Genomes in CMR– http://www.tigr.org/tigr-scripts/CMR2/CMR_

Content.spl


Genomes

– Fruit Fly– Mouse– Rat– Rice– Zebra fish– Puffer fish– Chicken– Dog– Frog


Growth of GenBank

• 1982: 600,000 Bases

• 2002: 28.5 Billion Bases


Other Notables

• Dayhoff ATLAS Database of Proteins (1960s)

• Sequence Comparison Algorithms– 1970, Needleman-Wunch (global alignment)

• Protein Databank– Brookhaven PDB (1973)


Other Notables

• NMR for protein structure identification (1980)

• IntelliGenetics Founded

– DNA and Protein sequence analysis (1980)


Other Notables

• Smith-Waterman algorithm– Local sequence alignment (1981)

• GenBank Database created (1982)

• Genetics Computer Group Founded– GCG suite (1982)

• PCR First Described (1985)


Other Notables

• FASTP Algorithm

– Protein database searching (1985)

• SWISS-PROT

– Protein Database (1986)


Other Notables

• PERL Programming Language– Allows for sequence manipulation (1987)

• NCBI Established (1988)

• Human Genome Initiative (1988)


Other Notables

• FASTA Program released (1988)– DNA and Protein sequence database searches

• BLAST Program released (1990)– Allows for quick database searches

• Informax Founded (1990)

• Human Genome Project Begins (1990)


Other Notables

• Creation and Use of ESTs Described (1991)

• Incyte Pharmaceuticals Founded (1991)

• TIGR Established (1992)

– Shotgun sequencing methods


Other Notables

• Affymetrix founded (1993)

• PRINTS protein motif database (1994)


Other Notables

First Commercial Microarray chips produced (1996)

• Dolly Cloned (1997)

• Capillary Sequencing machines introduced (1997)


Other Notables

• Celera Genomics Formed (1998)


More Detailed Histories

http://www.netsci.org/Science/Bioinform/feature06.html

http://www.dhgp.de/intro/history/history.html

3/17/2003 bioinformatics: merging biological and computational skills eric rouchka, d.sc. university...

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