CS 4233 5263 Bioinformatics Lectures 1 2 Introduction

CS 4233 & 5263 Bioinformatics Lectures 1 & 2: Introduction to Bioinformatics and Molecular Biology

Course description • A survey of algorithms and methods in bioinformatics, approached from a (more or less) computational viewpoint. • Prerequisite: – – Programming experience Strong background in algorithms and data structure Basic understanding of statistics and probability Appetite to learn some biology • For other information, check course website

Why bioinformatics • The advance of biomedical experimental technology has resulted in a huge amount of data – The human genome is “finished” – Even if it were, that’s only the beginning… • The bottleneck is how to integrate and analyze the data – Noisy – Diverse

Growth of Gen. Bank vs Moore’s law • Last week I just received 100 GB of DNA data from a “small” NGS experiment my collaborator did

Genome annotations Meyer, Trends and Tools in Bioinfo and Compt Bio, 2006

What is bioinformatics • National Institutes of Health (NIH): – Research, development, or application of computational tools and approaches for expanding the use of biological, medical, behavioral or health data, including those to acquire, store, organize, archive, analyze, or visualize such data.

What is bioinformatics • National Center for Biotechnology Information (NCBI): – the field of science in which biology, computer science, and information technology merge to form a single discipline. The ultimate goal of the field is to enable the discovery of new biological insights as well as to create a global perspective from which unifying principles in biology can be discerned.

Biology Molecular Biology Chemistry Medicine Bioinformatics Mathematics Statistics Physics Computer Science Informatics

Computer Scientists vs Biologists (courtesy Serafim Batzoglou, Stanford)

Biologists vs computer scientists • (almost) Everything is true or false in computer science • (almost) Nothing is ever true or false in Biology

Biologists vs computer scientists • Biologists seek to understand the complicated, messy natural world • Computer scientists strive to build their own clean and organized virtual world

Biologists vs computer scientists • Computer scientists are obsessed with being the first to invent or prove something • Biologists are obsessed with being the first to discover something

Some examples of central role of CS in bioinformatics

1. Genome sequencing AGTAGCACAGA CTACGACGAGA CGATCGTGCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3 x 109 nucleotides ~500 nucleotides

1. Genome sequencing AGTAGCACAGA CTACGACGAGA CGATCGTGCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3 x 109 nucleotides A big puzzle ~60 million pieces Computational Fragment Assembly Introduced ~1980 1995: assemble up to 1, 000 long DNA pieces 2000: assemble whole human genome

2. Gene Finding Where are the genes? In humans: ~22, 000 genes ~1. 5% of human DNA

2. Gene Finding 5’ Exon 1 Intron 1 Start codon ATG Exon 2 Intron 2 Splice sites Exon 3 3’ Stop codon TAG/TGA/TAA Hidden Markov Models (Well studied for many years in speech recognition)

3. Protein Folding • The amino-acid sequence of a protein determines the 3 D fold • The 3 D fold of a protein determines its function • Can we predict 3 D fold of a protein given its amino-acid sequence? – Holy grail of computational biology — 40 years old problem – Molecular dynamics, computational geometry, machine learning

4. Sequence Comparison—Alignment AGGCTATCACCTGACCTCCAGGCCGATGCCC TAGCTATCACGACCGCGGTCGATTTGCCCGAC -AGGCTATCACCTGACCTCCAGGCCGA--TGCCC--| | | | x | | | TAG-CTATCAC--GACCGC--GGTCGATTTGCCCGAC Sequence Alignment Introduced ~1970 BLAST: 1990, one of the most cited papers in history Still very active area of research Efficient string matching algorithms Fast database index techniques query DB BLAST

Lipman & Pearson, 1985 …, comparison of a 200 -amino-acid sequence to the 500, 000 residues in the National Biomedical Research Foundation library would take less than 2 minutes on a minicomputer, and less than 10 minutes on a microcomputer (IBM PC). Database size today (2007): 1012 (increased by 2 million folds). BLAST search: 1. 5 minutes

5. Microarray data analysis Example: Clinical prediction of Leukemia type • 2 types of leukemia – Acute lymphoid (ALL) – Acute myeloid (AML) • Different treatments & outcomes • Predict type before treatment? Bone marrow samples: ALL vs AML Measure amount of each gene

Some goals of biology for the next 50 years • List all molecular parts that build an organism – Genes, proteins, other functional parts • • • Understand the function of each part Understand how parts interact physically and functionally Study how function has evolved across all species Find genetic defects that cause diseases Design drugs rationally Sequence the genome of every human, use it for personalized medicine • Bioinformatics is an essential component for all the goals above

A short introduction to molecular biology

Life • Two main categories: – Prokaryotes (e. g. bacteria) • Unicellular • No nucleus – Eukaryotes (e. g. fungi, plant, animal) • Unicellular or multicellular • Has nucleus

Prokaryote vs Eukaryote • Eukaryote has many membrane-bounded compartment inside the cell – Different biological processes occur at different cellular location

Organism, Organ, Cell Organism n Orga

Chemical contents of cell • Water • Macromolecules (polymers) - “strings” made by linking monomers from a specified set (alphabet) –Protein –DNA –RNA –… • Small molecules –Sugar –Ions (Na+, Ka+, Ca 2+, Cl- , …) –Hormone –…

DNA • DNA: forms the genetic material of all living organisms – Can be replicated and passed to descendents – Contains information to produce proteins • To computer scientists, DNA is a string made from alphabet {A, C, G, T} – e. g. ACAGAACGTAGTGCCGTGAGCG • Each letter is a nucleotide • Length varies from hundreds to billions

RNA • Historically thought to be mainly an information carrier – DNA => RNA => Protein – Very important new roles have been found recently • To computer scientists, RNA is a string made from alphabet {A, C, G, U} – e. g. ACAGAACGUAGUGCCGUGAGCG • Each letter is a nucleotide • Length varies from tens to thousands

Protein • Protein: the actual “worker” for almost all processes in the cell – – – Enzymes: speed up reactions Signaling: information transduction Structural support Production of other macromolecules Transport • To computer scientists, protein is a string made from an alphabet of 20 letters – E. g. MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGP • Each letter is called an amino acid • Length varies from tens to thousands

DNA/RNA zoom-in • • Commonly referred to as Nucleic Acid DNA: Deoxyribonucleic acid RNA: Ribonucleic acid Found mainly in the nucleus of a cell (hence “nucleic”) • Contain phosphoric acid as a component (hence “acid”) • They are made up of a string of nucleotides

Nucleotides • A nucleotide has 3 components – Sugar ring (ribose in RNA, deoxyribose in DNA) – Phosphoric acid – Nitrogen base • • Adenine (A) Guanine (G) Cytosine (C) Thymine (T) in DNA and Uracil (U) in RNA

Units of RNA: ribo-nucleotide • A ribonucleotide has 3 components – Sugar - Ribose – Phosphate group – Nitrogen base • • Adenine (A) Guanine (G) Cytosine (C) Uracil (U)

Units of DNA: deoxy-ribo-nucleotide • A deoxyribonucleotide has 3 components – Sugar – Deoxy-ribose – Phosphate group – Nitrogen base • • Adenine (A) Guanine (G) Cytosine (C) Thymine (T)

Polymerization: Nucleotides => nucleic acids Nitrogen Base Phosphate Sugar

Free phosphate 5’ A G C 5 prime 3 prime 5’-AGCGACTG-3’ AGCGACTG G DNA A Often recorded from 5’ to 3’, which is the direction of many biological processes. e. g. DNA replication, transcription, etc. C T G 3’ 5 Phosphate 4 Base 1 Sugar 3 2

Free phosphate 5’ A G U 5 prime 3 prime 5’-AGUGACUG-3’ AGUGACUG G RNA A C U G 3’ Often recorded from 5’ to 3’, which is the direction of many biological processes. e. g. translation.

5’ A 3’ T G C C G G C A T C G T A G C 3’ 5’ Base-pair: A=T G=C Forward (+) strand 5’-AGCGACTG-3’ 3’-TCGCTGAC-5’ AGCGACTG TCGCTGAC Backward () strand One strand is said to be reversecomplementary to the other DNA usually exists in pairs.

DNA double helix G-C pair is stronger than A-T pair

Reverse-complementary sequences • 5’-ACGTTACAGTA-3’ • The reverse complement is: 3’-TGCAATGTCAT-5’ => 5’-TACTGTAACGT-3’ • Or simply written as TACTGTAACGT

Orientation of the double helix • Double helix is anti-parallel – 5’ end of one strand pairs with 3’ end of the other – 5’ to 3’ motion in one strand is 3’ to 5’ in the other • Double helix has no orientation –Biology has no “forward” and “reverse” strand –Relative to any single strand, there is a “reverse complement” or “reverse strand” –Information can be encoded by either strand or both strands 5’TTTTACAGGACCATG 3’ 3’AAAATGTCCTGGTAC 5’

RNA • RNAs are normally single-stranded • Form complex structure by selfbase-pairing • A=U, C=G • Can also form RNA-DNA and RNA -RNA double strands. – A=T/U, C=G

Protein zoom-in • Protein is the actual “worker” for almost all processes in the cell • A string built from 20 kinds of chars – E. g. MGDVEKGKKIFIMKCSQCHTVEKGGKH • Each letter is called an amino acid Side chain R | H 2 N--C--COOH | Carboxyl group Amino group H Generic chemical form of amino acid

Units of Protein: Amino acid • 20 amino acids, only differ at side chains – Each can be expressed by three letters – Or a single letter: A-Y, except B, J, O, U, X, Z – Alanine = Ala = A – Histidine = His = H

Protein R H 2 N R R R … N-terminal • • COOH C-terminal Has orientations Usually recorded from N-terminal to C-terminal Peptide vs protein: basically the same thing Conventions – Peptide is shorter (< 50 aa), while protein is longer – Peptide refers to the sequence, while protein has 2 D/3 D structure

Protein structure • Linear sequence of amino acids folds to form a complex 3 -D structure. • The structure of a protein is intimately connected to its function.

Genome and chromosome • Genome: the complete DNA sequences in the cell of an organism – May contain one (in most prokaryotes) or more (in eukaryotes) chromosomes • Chromosome: a single large DNA molecule in the cell – May be circular or linear – Contain genes as well as “junk DNAs” – Highly packed!

Formation of chromosome

Formation of chromosome 50, 000 times shorter than extended DNA The total length of DNA present in one adult human is the equivalent of nearly 70 round trips from the earth to the sun

Gene • Gene: unit of heredity in living organisms – A segment of DNA with information to make a protein or a functional RNA

Some statistics Chromosomes Bases Genes Human 46 3 billion 20 k-25 k Dog 78 2. 4 billion ~20 k Corn 20 2. 5 billion 50 -60 k Yeast 16 20 million ~7 k E. coli 1 4 million Marbled lungfish ? 130 billion ? ~4 k

Human genome • 46 chromosomes: 22 pairs + X + Y 1 from mother, 1 from father • Female: X + X • Male: X + Y

Human genome • Every cell contains the same genomic information – Except sperms and eggs, which only contain half of the genome • Otherwise your children would have 46 + 46 chromosomes …

Cell division: mitosis • A cell duplicates its genome and divides into two identical cells • These cells build up different parts of your body

Cell division: meiosis • A reproductive cell divides into four cells, each containing only half of the genomes – Diploid => haploid • Two haploid cells (sperm + egg) forms a zygote – Which will then develop into a multi-cellular organism by mitosis

Central dogma of molecular biology DNA replication is critical in both mitosis and meiosis

DNA Replication • The process of copying a double-stranded DNA molecule – Semi-conservative 5’-ACATGATAA-3’ 3’-TGTACTATT-5’

p p p Nucleotide triphosphate (d. NTP) • Mutation: changes in DNA base-pairs • Proofreading and error-correcting mechanisms exist to ensure extremely high fidelity

Central dogma of molecular biology

Transcription • The process that a DNA sequence is copied to produce a complementary RNA – Called message RNA (m. RNA) if the RNA carries instruction on how to make a protein – Called non-coding RNA if the RNA does not carry instruction on how to make a protein – Only consider m. RNA for now • Similar to replication, but – Only one strand is copied

Transcription (where genetic information is stored) DNA-RNA pair: A=U, C=G T=A, G=C (for making m. RNA) Coding strand: 5’-ACGTAGACGTATAGAGCCTAG-3’ Template strand: 3’-TGCATCTGCATATCTCGGATC-5’ m. RNA: 5’-ACGUAGACGUAUAGAGCCUAG-3’ Coding strand m. RNA have the same sequence, except that T’s in DNA are replaced by U’s in m. RNA.

Translation • The process of making proteins from m. RNA • A gene uniquely encodes a protein • There are four bases in DNA (A, C, G, T), and four in RNA (A, C, G, U), but 20 amino acids in protein • How many nucleotides are required to encode an amino acid in order to ensure correct translation? – 4^1 = 4 – 4^2 = 16 – 4^3 = 64 • The actual genetic code used by the cell is a triplet. – Each triplet is called a codon

The Genetic Code Third letter

Translation • The sequence of codons is translated to a sequence of amino acids • Gene: -GCT TGT TTA CGA ATT • m. RNA: -GCU UGU UUA CGA AUU • Peptide: - Ala - Cys - Leu - Arg - Ile – • Start codon: AUG – Also code Met – Stop codon: UGA, UAG

Translation • Transfer RNA (t. RNA) – a different type of RNA. – Freely float in the cell. – Every amino acid has its own type of t. RNA that binds to it alone. • Anti-codon – codon binding crucial. t. RNA-Pro Anti-codon Nascent peptide t. RNA-Leu m. RNA

Transcriptional regulation Transcription factor RNA Polymerase Transcription starting site promoter • • • gene Will talk more in later lectures RNA polymerase binds to certain location on promoter to initiate transcription Transcription factor binds to specific sequences on the promoter to regulate the transcription – Recruit RNA polymerase: induce – Block RNA polymerase: repress – Multiple transcription factors may coordinate

Splicing promoter Transcription starting site gene transcription Pre-m. RNA • Pre-m. RNA needs to be “edited” to form mature m. RNA • Will talk more in later lectures. intron Pre-m. RNA 5’ UTR exon 3’ UTR Splicing Mature m. RNA (m. RNA) Open reading frame (ORF) Start codon Stop codon

Summary • DNA: a string made from {A, C, G, T} – Forms the basis of genes – Has 5’ and 3’ – Normally forms double-strand by reverse complement • RNA: a string made from {A, C, G, U} – – – • Protein: made from 20 kinds of amino acids – – • m. RNA: messenger RNA t. RNA: transfer RNA Other types of RNA: r. RNA, mi. RNA, etc. Has 5’ and 3’ Normally single-stranded. But can form secondary structure Actual worker in the cell Has N-terminal and C-terminal Sequence uniquely determined by its gene via the use of codons Sequence determines structure, structure determines function Central dogma: DNA transcribes to RNA, RNA translates to Protein – Both steps are regulated

Experimental techniques to manipulate DNA

DNA synthesis • Creating DNA synthetically in a laboratory • Chemical synthesis – Chemical reactions – Arbitrary sequences – Maximum length 160 -200 • Cloning: make copies based on a DNA template – Biological reactions – Requires template – Many copies of a long DNA in a short time

Some terms • Denature: a DNA double-strand is separated into two strands – By raising temperature • Renature: the process that two denatured DNA strands re-forms a double-strand – By cooling down slowly • Hybridization: two heterogeneous DNAs form a double-stranded DNA – may have mismatches – The rationale behind many molecular biological techniques including DNA microarray

in vivo DNA Cloning • in vivo: in a living organism • Connect a piece of DNA to bacterial DNA, which can then be replicated together with the host DNA bacterial DNA

in vitro DNA Cloning • In vitro: outside of living organism • Polymerase chain reaction (PCR) 5’ 5’ denature 5’ 5’ Primer (< 30 bases) 5’ 5’ DNA Polymerase d. NTP 5’ 5’

DNA sequencing technology • Read out the letters from a DNA sequence • Chain-termination method (Sanger method) 1974, Frederick Sanger GTGAGGCGCTGC

DNA sequencing: Basic idea • PCR primer extension 5’-TTACAGGTCCATACTA 3’-AATGTCCAGGTATGATACATAGG-5’ • We need to supply A, C, G, T for the synthesis to continue • Besides A, C, G, T, we add some A*, C*, G*, and T* – Very similar to ACGT in all aspects, except that – The extension will stop if used

DNA sequencing, cont

Base calling

Sequencing speed • Current methods can directly sequence only relatively short (<1000 bp long) DNA fragments in a single reaction • Automated DNA-sequencing instruments (using gel-filled capillaries) can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a day: ~ 3, 000 bases per day

Advances in DNA sequencing • • 1969: three years to sequence 115 nt DNA 1979: three years to sequence ~1650 nt 1989: one week to sequence ~1650 nt 1995: Haemophilus genome sequenced at TIGR - 1, 830, 138 nt • 2000: Human Genome - working draft sequence, 3 billion bases • 2004: 454 Life Science invented the first new-generation sequencer

The bioinformatics landmark • Completion of human genome sequencing is a success embraced by – Advancement in sequencing technology – Speed of computation – Algorithm development in bioinformatics • HGP (Human Genome Project) strategy – Hierarchical sequencing – Estimated 15 years (1990 – 2005), completed in 13 years – $3 billion • Celera strategy – Whole-genome shotgun sequencing – Three years (1998 -2001) – $300 million

Prior to year 2007 • Over 300 genomes have been sequenced • ~1011 - 1012 nt

Year 2007 • Genomes of three individual human were sequenced – James Watson – Craig Venter – Yang Huanming • Cost for sequencing Watson’s genome – $3 million, 2 months – Compared to $3 billion, 13 years for HGP • These are achieved without the new-generation sequencing technology ! • June 3 2010: “Illumina Drops Personal Genome Sequencing Price to Below $20, 000”

• Sequencing speed has been tremendously improved • High efficiency and relatively low cost makes it possible to sequence the genome of any individual from any species What’s next?

Continue to sequence more species? Genome 10 K project More individuals? 1000 Genome project What to do with those sequences?

Coming next: biological sequence analysis