The document summarizes some of the complex challenges involved in genome analysis and sequencing. It discusses how genomes vary in size and features like sequence and polymorphism. Some major challenges are sequencing large chromosomes, distinguishing errors from true polymorphisms, and dealing with repeat sequences and unclonable DNA. Techniques like dividing chromosomes into overlapping clones that are sequenced multiple times can help meet these challenges. Linkage maps using markers like SNPs and SSRs help with genome-wide mapping, while physical maps integrate clones into contigs to span chromosomes. Sequencing strategies include hierarchical shotgun and whole genome shotgun approaches. The human genome project demonstrated improvements in sequencing capacity and integration of different map types. Insights from sequencing include the number of genes,

1. COMPLEX GENOME ANALYSIS Ikhide Imumorin, PhD Assistant Professor of Animal Molecular & Quantitative Genetics Cornell University Ithaca, NY 14853 USA

2. GENOMICS ANALYSIS

3. The genomes of living organisms vary enormously in size.

4. Genomicists look at two basic features of genomes: sequence and polymorphism. Major challenges to determine sequence of each chromosome in genome and identify many polymorphisms: How does one sequence a 500 Mb chromosome 600 bp at a time? How accurate should a genome sequence be? DNA sequencing error rate is about 1% per 600 bp. How does one distinguish sequence errors from polymorphisms? Rate of polymorphism in diploid human genome is about 1 in 500 bp. Repeat sequences may be hard to place. Unclonable DNA cannot be sequenced. Up to 30% of genome is heterochromatic DNA that can not be cloned

5. Divide and conquer strategy meets most challenges. Chromosomes are broken into small overlapping pieces and cloned. Ends of clones sequenced and reassembled into original chromosome strings Each piece is sequenced multiple times to reduce error rate. 10-fold sequence coverage achieves a rate of error less than 1/10,000.

6. Fig. 10.2

7. Techniques for mapping and cloning Cloning Library of DNA fragments 500 – 1,000,000 bp Insert into one of a variety of vectors Hybridization Location of a particular DNA sequence within the library of fragments PCR amplification Direct amplification of a particular region of DNA ranging from 1 bp to > 20kb DNA sequencing Automated DNA sequencer using Sanger method determines sequences 600 bp at a time. Computational tools Programs for identifying matches between a particular sequence and a large population of previously sequenced fragments Programs for identifying overlaps of DNA fragments Programs for estimating error rates Programs for identifying genes in chromosomal sequences

8. Making a large scale linkage map Types of DNA polymorphisms used for large-scale mapping: Single nucleotide polymorphisms (SNPs) – 1/500 – 1/1000 bp across genome Simple sequence repeats (SSRs) – 1/20-1/40 kb across genome 2-5 nucleotides is repeated 4-50 or more times. Most SNPs and SSRs have little or no effect on the organism. Serve as DNA markers across the chromosomes Must be able to rapidly identify and assay in populations from 100s to 1000s of individuals Fig. 10.3

9. Genome wide identification of genetic markers Initial genetic maps used SSRs which are highly polymorphic. Identified by screening DNA libraries with SSR probes Amplified by PCR and length differences assayed SNPs – millions more recently identified by comparison of orthologous regions of cDNA clones from different individuals

10. Homologous – genes with enough sequence similarity to be related somewhere in evolutionary history Orthologous – genes in two different species that arose from the same gene in the two species’ common ancestor Paralogous – arise by duplication within same species Orthologous genes are always homologous, but homologous genes are not always orthologous.

11. SNPs and SSRs for genome coverage Until recently, maps were constructed from about 500 SSRs evenly spaced across genome (1 SSR every 6 Mb). SNPs provide more than 500,000 DNA markers across the genome.

12. Genome wide typing of genetic markers Two-stage assay for simple sequence repeats PCR amplification Size separation Fig. 10.4

13. Long range physical maps: karyotypes and genomic libraries position markers on chromosomes. Physical map Overlapping DNA fragments ordered and oriented that span each of the chromosomes Based on direct analysis of DNA rather than recombination on which linkage maps are based Chart actual number of bp, kb, or Mb that separate a locus from its neighbors Linkage vs. physical maps 1 cM = 1 Mb in humans 1 cM = 2 Mb in mice

14. Vectors used for clone large inserts for physical mapping YACs (yeast artificial chromosomes) Insert size 100-1,000,000 Mb BACs (bacterial artificial chromosomes) Insert size 50 – 300 kb More stable and easier to purify from host DNA than YACs

15. How to determine order of clones across genome Overlapping inserts help align cloned fragments. Bottom-up approach – overlapping sequences of tens of thousands of clones determined by restriction site analysis or sequence tag sites (STSs) Top-down approach – insert is hybridized against karyotype of entire genome.

16. Identifying and isolating a set of overlapping fragments from a library Two approaches: Linkage maps used to derive a physical map Set of markers less than 1 cM apart Use markers to retrieve fragments from library by hybridization. Construct contigs – two or more partially overlapping cloned fragments. Chromosome walk by using ends of unconnected contigs to probe library for fragments in unmapped regions Physical mapping techniques: Direct analysis of DNA Overlapping clones aligned by restriction mapping Sequence tag segments (STSs)

17. Physical mapping by analysis of STSs Bottom-up approach Each STS represents a unique segment of the genome amplified by PCR. Fig. 10.5

18. Human Karyotype (a) Complete set of human chromosomes stained with Giemsa dye shows bands. (b) Ideograms show idealized banding pattern. Fig. 10.6 a, b

19. Chromosome 7 at three levels of resolution Fig. 10. 6 c

20. FISH protocol for top-down approach Fig. 10.8

21. Sequence maps show order of nucleotides in cloned piece of DNA. Two strategies for sequence human genome: Hierarchical shotgun approach Whole-genome shotgun approach Shotgun – randomly generated overlapping insert fragments: Fragments from BACs Fragments from shearing whole genome Shearing DNA with sonication Partial digestion with restriction enzymes

22. Hierarchical shotgun strategy Used in publicly funded effort to sequence human genome Shear 200 kb BAC clone into ~2 kb fragments Sequence ends 10 times Need about 1700 plasmid inserts per BAC and about 20,000 BACs to cover genome Data form linkage and physical maps used to assemble sequence maps of chromosomes Significant work to create libraries of each BAC and physically map BAC clones Fig. 10.9

23. Whole-genome shotgun sequencing Used by Celera Genomics to sequence whole human genome. Whole genome randomly sheared three times Plasmid library constructed with ~ 2kb inserts Plasmid library with ~10 kb inserts BAC library with ~ 200 kb inserts Computer program assembles sequences into chromosomes. No physical map construction Only one BAC library Overcomes problems of repeat sequences Fig. 10.10

24. Limitations of whole genome sequencing Some DNA can not be cloned. e.g., heterochromatin Some sequences rearrange or sustain deletions when cloned. Future large genome sequencing will use both shotgun approaches.

25. Sequencing of the human genome Most of draft took place during last year of project. Instrument improvements – 500,000,000 bp/day Automated factory-like production line generated sufficient DNA to supply sequencers on a daily basis. Large sequencing centers with 100-300 instruments – 150,000,000 bp/day

26. Integration of linkage, physical, and sequence maps Provides check on the correct order of each map against other two SSR and SNP DNA linkage markers readily integrated into physical map by PCR analysis across insert clones in physical map SSR, SNP (linkage maps), and STS markers (physical maps) have unique sequences 20 bp or more, allowing placement on sequence map.

27. Changes in biology, genetics and genomics from human genome sequence Genetics parts list Speeds gene-finding and gene-function analysis Sequence identification in second organism through homology Gene function in one organism helps understand function in another for orthologous and paralogous genes Genes often encode one or more protein domains Allows guess at function of new protein by comparison of protein sequence in databases of all known domains Ready access to identification of known human polymorphism Speeds mapping of new organisms by comparison e.g., mouse and human have high similarity in gene content and order

28. Major insights from human and model organism sequences Approximately 25,000 human genes Genes encode noncoding RNA or proteins. Repeat sequences are > 50% of genome. Distinct types of gene organization: Gene families Gene rich regions Gene desert Combinatorial strategies amplify genetic information and increase diversity. Evolution by lateral transfer of genes from one organism to another Males have twofold higher mutation rate than females. Human races have very few unique distinguishing genes. All living organisms evolve from a common ancestor.

29. Conserved segments of syntenic blocks in human and mouse genomes Fig. 10.12

30. Noncoding RNA genes Transfer RNAs (tRNAs) – adaptors that translate triplet code of RNA into amino acid sequence of proteins Ribosomal RNAs (rRNAs) – components of ribosome Small nucleolar RNAs (snoRNAs) – RNA processing and base modification in nucleolus Small nuclear RNAs (sncRNAs) - spliceosomes

31. Protein coding genes generate the proteome. Proteome – collective translation of 30,000 protein coding genes into proteins Complexity of proteome increase from yeast to humans. More genes Shuffling, increase, or decrease of functional modules More paralogs Alternative RNA splicing – humans exhibit significantly more Chemical modification of proteins is higher in humans.

32. Protein coding genes generate the proteome How transcription factor protein domains have expanded in specific lineages Fig. 10.11

33. Examples of domain accretions in chromatin proteins Fig. 10.13

34. Number of distinct domain architectures in four eukaryotic genomes Fig. 10.14

35. Repeat sequences fall into five classes. Transposon-derived repeats Processed pseudogenes SSRs Segmental duplications of 10-300 kb Blocks of repeated sequences at centromere, telomeres and other chromosomal features

36. Repeat sequences constitute more than 50% of the genome. Fig. 10.15

37. Gene Organization of Genome Gene families Closely related genes clustered or dispersed Gene-rich regions Functional or chance events? Gene deserts Span 144 Mb or 3% of genome Contain regions difficult to identify? e.g., big genes – nuclear transcript spans 500 kb or more with very large introns (exons < 1% of DNA)

38. Genome has a distinct organization. Gene family – olfactory receptor gene family

39. Class II region of human major histocompatibility complex contains 60 genes in 700 kb Fig. 10.17

40. Combinatorial strategies At DNA level – T-cell receptor genes are encoded by a multiplicity of gene segments. At RNA level – splicing of exons in different orders Fig. 10.19a Fig. 10.18

41. Lateral transfer of genes > 200 human genes may arise by transfer from organisms such as bacteria. Lateral transfer is direct transfer of genes from one species into the germ line of another.

42. Twofold higher mutation rate in males Comparison of X and Y chromosomes Same may be true for autosomes, but difficult to measure. Majority of human mutations arise in males. Males give rise to more defects, but also more diversity.

43. Human races have similar genes. Genome sequence centers have sequenced significant portions of at least three races. Range of polymorphisms within a race can be much greater than the range of differences between any two individuals of different races. Very few genes are race specific. Genetically, humans are a single race.

44. All living organisms are a single race. All living organisms have remarkably similar genetic components. Life evolved once and we are descendents of that event. Analysis of appropriate biological systems in model organisms provides fundamental insight into corresponding human systems.

45. In the future, other features of chromosomes will become increasingly important. Chemical modification of bases Understand DNA methylation now Others may be discovered Interaction of various proteins with chromosome Three dimensional structure of proteins in nucleus May determine interactions of chromosomal regions with regions of nuclear envelope More effective tools need to be developed to examine chromosome features.

47. High-throughput instruments DNA sequencer Fig. 10.20

48. High-throughput instruments e.g, microarrays Fig. 10.21

49. Two color - DNA microarray Fig. 10.22

50. Analysis of genomic and RNA sequences Quantitative analysis of mRNA levels Serial analysis of gene expression (SAGE) Small cDNA tags of 15 bp from 3’ ends of mRNA are linked and sequenced. Massively parallel signature sequence (MPSS) Transcriptome – population of mRNAs expressed in a single cell or cell type MPSS allows identification of most of cell’s rarely expressed mRNAs

51. Lynx therapeutics sequencing strategy of MPSS Fig. 10.24

52. Systems Biology – the global study of multiple components of biological systems and their interactions New approach to studying biological systems has made possible: Sequencing genomes High-throughput platform development Development of powerful computational tools The use of model organisms Comparative genomics

53. Human Genome Project has changed the potential for predictive/preventive medicine. Provided access to DNA polymorphisms underlying human variability Makes possible identification of genes predisposing to disease Understanding of defective genes in context of biological systems Circumvent limitations of defective genes Novel drugs Environmental controls Approaches such as stem-cell transplants or gene therapy

54. Social, ethical, and legal issues Privacy of genetic information Limitations on genetic testing Patenting of DNA sequences Society’s view of older people Training of physicians Human genetic engineering Somatic gene therapy – inserting replacement genes Germ-line therapy – modifications of human germ line