Transcript Human Genome

Human Genome Lecture
• Historical aspects of the HGP
• EST sequencing:
– Finding new genes faster than ever
• Using 3’ ESTs to generate human gene
maps
– First comprehensive genome-wide human gene
maps
• Sequence of human genome
• Complex genomic regions and sequence
limitations
Key pre-HGP scientific advances
• Structure of DNA determined (1953)
– Watson & Crick
• Recombinant DNA created (1972)
– P. Berg; Cohen and Boyer
• Methods for DNA sequencing developed (1977)
– Maxam & Gilbert; F. Sanger
• PCR invented (1985)
– K. Mullis
• Automated DNA sequencer developed (1986)
– L. Hood
Obstacles to formation of the HGP
1) Financial/political: “Big biology is bad biology”
-departure from cottage industry culture of biology
-devoid of “hypothesis-driven” research
-what will it cost?
-will it take away $$ from other programs?
2) Why sequence the “Junk”?
-protein coding regions make up <1.5% of the genome
-waste of time/money to sequence repetitive, hard-tosequence regions
3) It is impossible to do
-mid 1980s:
-primitive sequencing capabilities (500 bp/day/lab)
-primitive computer capabilities/bioinformatics
resources
Significance of the HGP
• “The book of life”, “The grail of human biology”, “Code
of codes”
• The instructions to create a human being
• The genome is a product of evolution
- molecular replicator (DNA) + heritable variation +
time + changing environment = genome
- record of the evolutionary history of our species
• Comparative genomics & “the genes that make us
human”
• The genome: unparalled system of information storage
- 70 trillion cells in human body
- each cell stores 3 billion units of information
Significance of the HGP (cont)
• Biology in the 21st century:
- equivalent of learning to read a new language
• The genome as dynamic not static:
- perspective on past/future of the species
• Implications for health and disease:
-Genetic disease gene discovery: single-gene
diseases & multifactorial diseases
-DNA-based diagnostics
-New drug targets
-Gene therapy implications
-Therapeutic uses vs. enhancements
• Accumulation of a molecular “parts lists” of human
physiology & anatomy:
- Lander: “Periodic Table of the Elements” analogy
Genomics Timelines
Rapid Gene Identification & Mapping:
ESTs and Gene-based STSs
• Single-pass sequencing of randomly selected cDNA
clones
• Obtain sequences from 5’ and 3’ ends of cDNA
inserts
• Rapidly & cheaply identify human genes
– Alzheimer’s gene discovered by EST database search
• 3’UT sequence: ideal for STS development & PCRbased gene mapping
• Readily scaled up for development of most
comprehensive human gene maps (Science 1996,
1998)
“One gene = one STS”
• Gene-based STSs as the basis for a
human gene map
– Berry et al, Nature Genetics 1995
• ESTablishing a human transcript map
– Boguski and Schuler, Nature Genetics
1995
Boguski & Schuler, Nat. Genet. 1995
Size and gene
content of the 24
human
chromosomes.
A, Size of each human
chromosome, in millions of
base pairs (1 million base
pairs = 1 Mb).
Chromosomes are ordered
left to right by size. B,
Number of genes
identified on each human
chromosome.
Chromosomes are ordered
left to right by gene
content. (Based on
www.ensembl.org, v36.)
Genomic sequencing vs EST sequencing
• EST (single pass cDNA) sequencing
- very fast but not error-free (e.g. 99% accuracy)
- very rapid gene identification (reliance on mRNA)
- cDNA abundance influences coverage
some genes will be missed
normalized cDNA libraries improve coverage
provides a gene expression profile
• Genomic sequencing
- pre-2001: much slower method for gene finding
-must do gene id by computer prediction
- will generate complete gene and genome information, e.g.
introns, regulatory regions, intergenic regions, repeats,
etc.
- more expensive way to id genes
- independent of gene expression level concerns
- highly accurate when complete
Table 11. Genome overview.
Size of the genome (including gaps)
Size of the genome (excluding gaps)
Longest contig
Longest scaffold
Percent of A+T in the genome
Percent of G+C in the genome
Percent of undetermined bases in the genome
Most GC-rich 50 kb
Least GC-rich 50 kb
Percent of genome classified as repeats
Number of annotated genes
Percent of annotated genes with unknown function
Number of genes (hypothetical and annotated)
Percent of hypothetical and annotated genes with unknown function
Gene with the most exons
Average gene size
Most gene-rich chromosome
Least gene-rich chromosomes
Total size of gene deserts (>500 kb with no annotated genes)
Percent of base pairs spanned by genes
Percent of base pairs spanned by exons
Percent of base pairs spanned by introns
Percent of base pairs in intergenic DNA
Chromosome with highest proportion of DNA in annotated exons
Chromosome with lowest proportion of DNA in annotated exons
Longest intergenic region (between annotated + hypothetical genes)
Rate of SNP variation
2.91 Gbp
2.66 Gbp
1.99 Mbp
14.4 Mbp
54
38
9
Chr. 2 (66%)
Chr. X (25%)
35
26,383
42
39,114
59
Titin (234 exons)
27 kbp
Chr. 19 (23 genes/Mb)
Chr. 13 (5 genes/Mb), Chr. Y (5 genes/Mb)
605 Mbp
25.5 to 37.8*
1.1 to 1.4*
24.4 to 36.4*
74.5 to 63.6*
Chr. 19 (9.33)
Chr. Y (0.36)
Chr. 13 (3,038,416 bp)
1/1250 bp
In these ranges, the percentages correspond to the annotated gene set (26, 383 genes) and the hypothetical + annotated gene set
(39,114 genes), respectively.
*
Significant findings arising from analysis of the draft
sequence of the human genome
• The genomic landscape shows marked variation in the distribution of a number of features, including genes,
transposable elements, GC content, CpG islands and recombination rate. This gives us important clues about function.
For example, the developmentally important HOX gene clusters are the most repeat-poor regions of the human genome,
probably reflecting the very complex coordinate regulation of the genes in the clusters.
• There appear to be about 30,000–40,000 protein-coding genes in the human genome—only about twice as many as in
worm or fly. However, the genes are more complex, with more alternative splicing generating a larger number of protein
products.
• The full set of proteins (the 'proteome') encoded by the human genome is more complex than those of invertebrates.
This is due in part to the presence of vertebrate-specific protein domains and motifs (an estimated 7% of the total), but
more to the fact that vertebrates appear to have arranged pre-existing components into a richer collection of domain
architectures.
• Hundreds of human genes appear likely to have resulted from horizontal transfer from bacteria at some point in the
vertebrate lineage. Dozens of genes appear to have been derived from transposable elements.
• Although about half of the human genome derives from transposable elements, there has been a marked decline in the
overall activity of such elements in the hominid lineage. DNA transposons appear to have become completely inactive
and long-terminal repeat (LTR) retroposons may also have done so.
• The pericentromeric and subtelomeric regions of chromosomes are filled with large recent segmental duplications of
sequence from elsewhere in the genome. Segmental duplication is much more frequent in humans than in yeast, fly or
worm.
• Analysis of the organization of Alu elements explains the longstanding mystery of their surprising genomic distribution,
and suggests that there may be strong selection in favour of preferential retention of Alu elements in GC-rich regions and
that these 'selfish' elements may benefit their human hosts.
• The mutation rate is about twice as high in male as in female meiosis, showing that most mutation occurs in males.
• Cytogenetic analysis of the sequenced clones confirms suggestions that large GC-poor regions are strongly correlated
with 'dark G-bands' in karyotypes.
• Recombination rates tend to be much higher in distal regions (around 20 megabases (Mb)) of chromosomes and on
shorter chromosome arms in general, in a pattern that promotes the occurrence of at least one crossover per
chromosome arm in each meiosis.
• More than 1.4 million single nucleotide polymorphisms (SNPs) in the human genome have been identified. This
collection should allow the initiation of genome-wide linkage disequilibrium mapping of the genes in the human
population.
Patterns of intrachromosomal and interchromosomal
duplication in the human genome
Bailey, et al, Science, 2002
HapMap phase 1
Distribution of >50 kb gaps in
HapMap phase 1 - CEU
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
0
base position (Mb)
50
100
150
200
chromosome lengths
>50 kb gap between SNPs
excluding centromere gaps
heterochromatin
250
T. Hudson
Bailey, et al, Science, 2002
Genome “Structural Variation”
• Broadest sense: all changes in the
genome not due to single base-pair
substitutions:
• Copy number variations (CNVs)
– CNV loci may cover 12% of genome
• Insertions/Deletions (indels)
– e.g. Repeats: STRs, VNTRs
• Inversions
• Duplications and translocations
Limitations of Genome Sequencing
• Nexgen sequencers are short read
– Repeated/duplicated sequences often can’t be positioned
• Segmental duplications make up 5% of genome
– >95% identity; >20kb
– Smaller-size, highly duplicated sequence families exist
• Complex, duplication-rich regions
–
–
–
–
–
>200 gaps (>50kb each) in human genome
Difficult to accurately assemble
Linked to many human diseases
Linked to evolutionary adaptation
Location of “missing heritability” of GWAS?
• Are critical regions of the genome being missed/ignored?
Limitations of next-generation genome
sequence assembly
Can Alkan, Saba Sajjadian & Evan E Eichler
Nature Methods Volume: 8, Pages: 61–65 Year published: (2011) DOI:
doi:10.1038/nmeth.1527 Published online 21 November 2010 Abstract
Abstract
High-throughput sequencing technologies promise to transform the
fields of genetics and comparative biology by delivering tens of
thousands of genomes in the near future. Although it is feasible to
construct de novo genome assemblies in a few months, there has been
relatively little attention to what is lost by sole application of short
sequence reads. We compared the recent de novo assemblies using the
short oligonucleotide analysis package (SOAP), generated from the
genomes of a Han Chinese individual and a Yoruban individual, to
experimentally validated genomic features. We found that de novo
assemblies were 16.2% shorter than the reference genome and that
420.2 megabase pairs of common repeats and 99.1% of validated
duplicated sequences were missing from the genome. Consequently, over
2,377 coding exons were completely missing. We conclude that highquality sequencing approaches must be considered in conjunction with
high-throughput sequencing for comparative genomics analyses and
studies of genome evolution.