The Human Genome at Ten

Transcription

1 The Human Genome at Ten Jutta Marzillier, Ph.D Lehigh University Biological Sciences December 3 rd, 2010

2 The Human Genome at 10 How did it start, how was it done, and what is its Impact on Science and Medicine? Jutta Marzillier, Ph.D Lehigh University Biological Sciences December 3 rd, 2010

3 10 years ago in June 2000 at the White House: First rough draft of the book of life announced Clinton: Completion of the Genome Project would revolutionize the diagnosis, prevention and treatment of most, if not all, human diseases Craig Venter Bill Clinton Francis Collins Collins: personalized medicine is likely to emerge by the year

4 10 years ago at the White House: First rough draft of the book of life announced 2010: The next 10 years Craig Venter Bill Clinton Francis Collins

5 Year of Jan Feb Feb 2010 the genomic Era.. 4 Mar Apr Apr May 2010

6 Outline Rationale for Genome Sequencing Method of DNA sequencing DNA sequencing technologies Major insights and achievements What s next?

7 Rationale: Why is the Knowledge about the Human Genome interesting? The human body has about 100 trillion cells. Each cell harbors the same genetic information in its nucleus in form of DNA containing chromosomes. Depending on cellular, developmental, and functional stage of a cell only a subset of genes is expressed. Modifications in the genetic information may result in disease.

8 Why is Genome Sequencing Important? To obtain a blueprint DNA directs all the instructions needed for cell development and function To study gene expression in a specific tissue, organ or tumor To study how humans relate to other organisms DNA underlies almost every aspect of human health, both, in function and dis-function To find correlations how genome information relates to development of cancer, susceptibility to certain diseases (personalized medicine) and drug metabolism (pharmacogenomics)

9 What is DNA? DNA constitutes the heritable genetic information that forms the basis for the developmental programs of all living organisms. A genome is an organism s complete set of DNA Watson & Crick, 1953 The DNA is made up of four building blocks called nucleotides. DNA sequence is the particular side-by-side arrangement of bases along a DNA strand. DNA sequencing is a biochemical method to determine the sequence of the nucleotide bases that make up the DNA.

10 Method of DNA sequencing: Principle of DNA Synthesis Arthur Kornberg demonstrated DNA replication in a cell-free (in vitro) bacterial extract (Nobel prize, 1959) -Discovered DNA polymerase (Pol1) to facilitate t DNA synthesis - Unraveled the mechanism of DNA synthesis - building blocks (Adenine, Cytosine, Guanine, Thymine) - a single DNA strand serves as a template - can only extend a pre-existing chain (primer) - a free 3 Hydroxyl end is required Watson et al., MGB, 2008

11 Reaction Mechanism for DNA synthesis The 3 hydroxyl group of the primer attacks the -phosphoryl group of the incoming nucleotide thereby forming a phosphodiester bond (S N 2 reaction). Watson et al., MBG, 2008

12 Dideoxy Sequencing according to Sanger Frederick Sanger Nobel Prize (1980) Both nucleotide types can be incorporated into growing DNA chain. Presence of dideoxy-cytosine in growing chain blocks further addition of fincoming i nucleotides. Watson et al., MBG, 2008

13 Dideoxy Method of Sequencing (Sanger, 1975) Normal DNA synthesis: DNA strand as template Primer Deoxynucleotides Polymerase enzyme Use several cycles to amplify DNA sequencing DNA synthesis is carried out in the presence of limiting amounts of dideoxyribonucleoside triphosphates that results in chain termination Through chain termination fragments of distinct sizes are generated that can be separated by gel electrophoresis Original method used radio- GCTACCTGCACCA labeled primers or GCTACCTGCA dideoxynucleotides GCTACCTGCACCAGA GCTA

14 Dideoxy Method of Sequencing Original method used radio-labeled primer or dideoxynucleotides This method required four separate DNA synthesis reactions to be separated by electrophoresis in four parallel lanes. The gel needs to be dried, exposed to film, developed and manually read. Approx bases read length Summary Sequencing Method Established - Need of four reactions in parallel - Heat labile polymerase - Use of radioactivity - Low resolution on gels - Approx. 150 nucleotides read length - time consuming Improvements -Use of fluorescently labeled dideoxy- nucleotides -One-lane electrophoresis -Introduction of capillary electrophoresis to increase resolution (up to 1,000 ntes) -Use of heat stable polymerase (Taq) - Automation

15 Fluorescently labeled ed ddntps used for sequencing reaction allow one lane analysis ddntps used for automated sequencing are labeled with different fluorescent dyes representing each of the 4 bases dnaseq.genomics.com.cn/dnaseq/en/tech/images Allows separation of DNA products in same lane of gel

16 Capillary Gel Electrophoresis Increases Resolution Sequence ladder by radioactive sequencing compared to fluorescent peaks Capillary gel electrophoresis: Samples passing a detection window are excited by laser and emitted fluorescence is read by CCD camera. Fluorescent signals are converted into basecalls. Advantage g High resolution Read length up to 1,000 nucleotides

17 ABI 310 Genetic Analyzer One-capillary Instrument Capillary and electrode: Negatively charged DNA migrates towards anode DNA fragments labeled with different fluorescent dyes migrate according to their size past a laser

18 Sequencing Electropherogram Laser excites dyes, causing them to emit light at longer wavelengths Emitted light is collected by a CCD camera Software converts pattern of emissions into colored peaks Plot of colors detected in sequencing sample scanned from smallest to largest fragment

19 Finishing Draft Sequences using Assembly g g y Software Primary sequencing reads:

20 Finishing Draft Sequences using Assembly Software Primary sequencing reads: Align sequences for homologies Green: 3-fold coverage Yellow: 2-fold coverage Fragment assembly to contig Goal for Genome Sequencing: Sanger: 8-fold coverage 454: 30 fold coverage

21 Sequencing Technologies: Life Sciences Thrive Trough their Co-laboration with Engineering and BioInformatics Biologist BioInformatics Engineers ww ww.itb.cnr.it

22 How is Genome Sequencing Done? Clone by Clone Create a crude physical map of the whole genome by restriction mapping before sequencing Shotgun sequencing Break genome into random fragments, sequence each of the fragments and assemble fragments based on sequence overlaps Break the genome into overlapping fragments and insert them into BACs and transfect into E.coli Break these inserts in even smaller fragments and sequence

23 High-Throughput Whole Genome Sequencing Analysis of 384 sequencing reactions in parallel George Church, Scientific American, January 2006, pp47-54

24 JGI Sequencing Facility (Joint Genome Institute, US Department of Energy) Assume more than capillary sequencers running simultaneously approx. 700 bp per capillary run approx. 3 hours per run Approx 40 Million bases per day Approx. 40 Million bases per day in one facility

25 What Challenge Was it to Sequence the Human Genome? It took more than 6 years to determine the complete base sequence of the E. coli genome (4.6 Mb). The Human Genome has 3200 Mb. At this rate it would have taken a single lab more than 4,000 years to sequence the entire human genome The average fragment read length is about 500 bases. It would take a minimum of six million (3 billion/500) to sequence the human genome (no overlap, 1-fold coverage). Develop procedural and computational algorithms and efficient database management International Co-laboration effort started Initial sequencing and analysis of the human genome Nature 409, (15 February 2001) International Human Genome Sequencing Consortium(headed by Francis Collins) The Sequence of the Human Genome, Science, Vol 291, Issue 5507, , 16 February 2001 Venter et al. (Celera Genomics)

26 In 2001 the First Draft of the Human Genome is Published How many nucleotide pairs does the Human Genome have? 3 x 10 6 or 3 x 10 9? How many genes does it take to make a human? Roundworm: 6,000 Fruitfly: 14,000 How different are genomes from one individual to another? 0.1% or 1% or 10 %?

27 What did we learn? Less than 2% of the Human Genome codes for protein The human genome encodes for approx. 21,000 protein-coding genes The human genome sequence is almost exactly the same (99.9%) 9%) in all people Non-coding DNA segments may have regulatory functions on gene expression The genome size does not correlate with the number of estimated genes Organism Genome Size Est. Gene # E. coli 4.6 Mb 4,400 Yeast 12.1 Mb 6,200 Humans have only about twice the number of genes as a fruit fly and barely more genes than a worm! Roundworm 97 Mb 19,700 Fruit Fly 180 Mb 13,600 Rice 389 Mb 37,500 Human 3200 Mb 25,000

28 Who s sequence was used for the Human Genome Project Different sources of DNA were used for original sequencing Celera: 5 individuals; HGSC: many The term genome is used as a reference to describe a composite genome The many small regions of DNA that vary among individuals are called polymorphisms: Mostly single nucleotide polymorphisms (SNPs) Insertions/ deletions (indels) Copy number variation Inversions genetic_variation_i_what_is_a.php#more SNPs: the human genome has at least 20 million SNPs most of these SNPs contribute to human variation some of them may influence development of diseases, susceptibility to certain drugs, toxins, infectious agents

29 Development Sequencing Time Line 1953: Discovery of the DNA double helix by Watson & Crick

30 The Race for the $1,000 Genome Human Genome Project (2001, initial draft): > $ 3 billion (includes development of technology) raw expenses estimated at $300 million Rhesus macaque (2006) $ 22 million By end of 2007: $ 1-2 million for full mammalian genome sequence Wanted:!!!!!! The $ 1,000 Genome!!!!!!!!! - low cost - high-throughput -high accuracy

31 454 Pyrosequencing Method Flowgram: Add one kind of dntp per cycle. Sulfurylase converts PPi to ATP in presence of APS Luciferin conversion results in light emission Problem: homopolymer detection Intensity of light emission is directly Proportional to amount of released PPi molecules l Sequencing is recorded live. Read length up to 400 bases

32 Genome Sequencing Milestones May 2007: James Watson s genome deposited 454 technology (pyrosequencing) 2 month,,$ 1-2 million Sept 2007: Craig Venter s genome deposited Sanger technology, 4 years, 70 million *Diploid genomes Appliedbiosystems.com *Approx. 3.5% of Watson s genome could not be matched to the reference genome *Venter s genome had 4.1 million DNA variants comprising 12.3 Mb million SNPs - non-snp variants Personal Genome Project (PGP): initiated by George Church (Harvard) seeking for volunteers to share their DNA sequence and medical records

33 Next Generation Sequencing (NGS) Focuses on Miniaturization and Parallelization Sanger Cyclic Array Sequencing Generate dense planar array of DNA features Apply cycles of enzymatic driven biochemistry Imaging-based data collection

34 Sequence Mate-Pairs Provide an Additional Layer of Positional Information DNA fragmentation in nebuilizer, end polishing, size selection by DNA gel electrophoresis Adapter ligation and sequencing Sequence mate-pairs separated by a known distance Assemble contigs into larger scaffolds using information about sequence similarity and distance between mate-pairs

35 Next generation sequencing 454 method (Pyrosequencing) -Shear DNA into 300 to 400 nte fragments -Ligate to heterobifunctional adapters -Capture DNA strands on beads and amplifiy by emulsion PCR -Place beads with amplified DNA fragments into picotiter plate (1.7 Mio wells) - Perform pyrosequencing reaction 44 m

36 Solexa/ Illumina Sequencing Technology Illumina reversible Sanger reversible Use a set of deoxynucleotides that carry - each a fluorescent label that can be cleaved off - a reversibly terminating moiety at the 3 hydroxyl position

37 Solexa/ Illumina Sequencing Technology

38 Comparison Sanger Sequencing: In vitro construction of library Limited parallelization large volumes (microliter) Expensive 454 Sequencing: Direct in-vitro amplification Massively parallel Reduction of reagent volume to pico or femtoliters per DNA feature Relatively l cheap Long reads (up to 1000 bases) High accuracy (0.1% error) 4 x 10 5 bases/run Shorter read length ( bases) Lower raw accuracy (1% error) 6 x 10 8 bases/run Illumina sequencing Read length bases 2 x 10 9 bases/run

39 BGI The Sequencing Factory (Beijing Genome Institute) Featured in Nature issue March 4 th, 2010 Purchased 128 HiSeq2000 sequencers encers from Illumina in January ar 2010 each of which can produce 25 billion base pairs of sequence a day

40 Speed Reading 1987: ABI sequencer 4800 basepairs a day 2010: Life Technologies 100 billion basepairs a day C. Venter, nature, 464, pg676, 2010

41 All published fully sequenced human genomes since 2000

42 Outlook: Single Molecule Sequencing through Graphene nanopore Graphene (Geim & Novoselov, Nobel Prize 2010) is a planar sheet of carbon just one atom thick Strongest material known, electrically conductive As a DNA chain passes through the nanopore, the nucleobases, which are the letters of the genetic code, can be identified. The nanopore in graphene is the first nanopore short enough to distinguish between two closely neighboring nucleobases

43 Achievements: The duck-billed platypus: part bird, part reptile, part mammal and the genome to prove it - The Platypus is our most distant t mammal relative - Has retained a large overlap between two very different classifications - The platypus shares 82% of its genes with human, mouse, dog, opossum and chicken - Decoding the platypus genome helps to understand the origins of mammal evolution Nature, May 7, 2008

44 Decoding of a Acute Myeloid Leukemia Genome * AML is a cancer of white blood cells, which h accumulate in the bone marrow and interfere with the production of normal white and red blood cells. * Sequenced and compared the genome of cancerous bone marrow cells with normal skin cells from the same patient using the next generation technologies. * Found a total t of 10 genes carrying mutations ti in the cancerous cells. * Two of those (FLT3 and NPM1) had already been implicated in the process of progression to AML. Proof of concept that whole genome sequencing of ftumor cells is a very useful strategy t to uncover genes implicated in the disease. Ley et al., nature 456:66, 2008

45 Twin study surveys genome for cause of Multiple Sclerosis MS causes the body s own immune cells to attack the myelin sheath around nerve cells MS has a genetic component No clear genetic reason found to explain why one twin developed MS while the other did not Both twins carry genetic variants that are linked to a higher risk getting MS Baranzini et al. April 29 th, 2010 But those genetic factors seem to have been insufficient to cause disease of their own Environmental triggers, epigenetics?

46 Common Gene Variant Strategy Assumption: To predict human diseases or susceptibility common DNA variations (~5% frequency) would be at a fault. Many SNP alleles have been uncovered related to specific diseases. however, these variants have only accounted for a small fraction of disease risk. A gene is vexed with multiple layers of complexity (Joseph Nadeau) Hall. Revolution Postponed. Scientific American, Oct. 2010, pg 60-67

47 In Contrast: Look at Rare Variants and Beyond the Genes Other mechanisms of gene expression regulation: Inhibitory RNA (RNAi) Chemical DNA modification, e.g. methyl groups

48 Lessons learned Data sharing 2000: 4 eukaryotic genomes 2010: 250 eukaryotic genomes, many hundreds of human genomes Fewer protein-coding genes than thought Studying of genetic variation and evolutionary origins Cost of sequencing has fallen 100,000 fold; high trough-put sequencing HapMap project: Catalog more than 20 million SNPs Cancer genome atlas to map genomic changes observed in every major type of human cancer Obtaining a genome blueprint may be sufficient to explain the susceptibility to disease,, but not the likelyhood to develop the disease. The combination of m a n y factors needs to be taken into account

49 Ethic Aspects of Genome Sequencing nhs.needham.k12.ma.us -How can patient privacy be protected? -Will insurance companies and employers use genetic information to screen out those at high risk for disease? -Genetic engineering of bioterrorism agents

50 What s next? Conference Topics: Genetic basis of common diseases Intricacies of gene regulation Role of non-coding portions of the genome Medical advances Technological and ethical challenges of human genomics Personalized genomics Aspects of variation in the human genome