Genomic structural variation

Transcription

1 Genomic structural variation Mario Cáceres The new genomic variation DNA sequence differs across individuals much more than researchers had suspected through structural changes A huge amount of structural variation has been discovered in the genome of all the studied species, which could have a much bigger biological impact than SNPs 1

2 Overview 1. Types of structural variants (SVs) 2. Methods for detecting SVs 3. Indels and CNVs 4. Inversions 5. Mechanisms of generation 6. Functional effects and examples What are structural variants Genomic alterations other than nucleotide substitutions that change the organization of the DNA molecule Traditionally detection limited by resolution of techniques and considered to be quite large Currently include changes at the base level, although typically are >1kb (no clear size limit) 2

3 Types of structural variants A B C D Reference A B C E D Insertion A B C Deletion CNVs A B C C C D Copy Number Variation A C B D Inversion and traslocations. Methods for detecting SVs New genomic techniques have changed the focus from large to small events and from locus-specific to global studies: Cytogenetic techniques (regular karyotyping, FISH, fiber FISH) Array comparative genomic hybridization (acgh) (BAC arrays, high-density oligo arrays) SNP array genotyping (Afymetrix, Illumina) Pair-end mapping (Sanger or next-generation sequencing) + Throughput + Resolution 3

4 Cytogenetic techniques Karyotyping FISH Chromosome painting Fiber FISH Array CGH Ratio of fluoresecence intensity of the test and the reference DNA indicates the differences in copy number for a particular location in the genome 4

5 Characteristics of acgh arrays BAC arrays High-density oligonucleotide arrays 1 BAC ( kb) per genomic region (Whole-genome tiling path) Good genomic coverage Low resolution (>20-50 kb) Variable oligo spacing (~2-10 kb on Agilent 244K or 1M arrays) Targeted or whole-genome approach Coverage depending on probe density High resolution ( kb) Pair-end mapping (PEM) method High-throughput way of detecting all kind of medium and large sized SVs from an individual: 1. Generation of DNA library of fragments of defined size from sample of interest 2. Sequencing of both ends of a large number of fragments 3. Mapping of both ends to reference genome and computational prediction of SVs 5

6 Pair-end mapping (PEM) methods PEM distribution Normally mapped PEM span > cutoff D Opposite end orientation PEM span < cutoff I Short-fragment PEM: Low coverage/high resolution (500 bp - 3 kb) Long-fragment PEM: High coverage/low resolution (10 kb 40 kb) SV detection methods comparison Feuk et al. Nature Reviews Genetics 7, (February 2006) doi: /nrg1767 6

7 Importance of structural variants Total entries: 89,427 CNVs: 57,829 Inversions: 850 InDels (100 bp - 1 kb): 30,748 Total CNV loci: 14,478 Articles cited: 38 (march 25, 2010) Segmental duplications Inversion breakpoints Copy number variants Timescale of CNVs study (Beckman et al., Nat. Rev. Genet. 2007) CNVs discovery is not that recent! 7

8 Initial large-scale CNV studies Analysis of 20 individuals using low density arrays 76 unique CNV regions of >100 kb ~11 CNVs differing between pair of individuals Analysis of 55 individuals using BAC arrays (1 Mb resolution) 224 unique CNV regions 12.4 CNVs differing between pair of individuals Global analysis of human CNVs CNV map by WGTP BAC arrays and Affymetrix arrays 270 individuals of European, African and Asian ancestry 1447 CNV regions covering 360 Mb (12% of the genome) Comprenhensive CNV map by tiling oligonucleotide arrays (42 million probes) 450 individuals of European, African and Asian ancestry CNVs assayed of >0.5 kb 8

9 Comprehensive human CNV map Overview of Experimental strategy for CNV discovery and genotyping (Conrad et al., Nature 2009) On average: 1098 validated CNVs differ between two individual genomes Detected CNVs ranged from 443 bp to 1.28 Mb (median size 2.9 kb) Validated CNVs cover a total of Mb (3.7% of the genome) Population distribution of CNVs CNV-based phylogenetic tree CNVs across 29 human populations 9

10 CNVs in other species CNVs in other species 10

11 Human polymorphic inversions Chrom. region Size Freq. Detection method Phenotypic effect Origin Reference Xq28 49 Kb 8-72% Southern unknown NAHR Small q Mb 5% FISH/PFGE Williams susceptibility? NAHR Osborne p Mb 15% FISH unknown NAHR? Giglio p Mb 9% FISH unknown NAHR? Giglio q Mb 4% FISH/PFGE unknown - Gimelli p Kb - Comp. map. unknown - Martin q Kb 17% PFGE/genot Increased fertility NAHR Stefansson p q Mb - Cloning unknown DSB? Gilling 2006 Insights from genomic studies 11

12 Insights from genomic studies New genomic techniques have uncovered an unprecedented degree of all types of structural variation, including inversions: Feuk et al., PLOS Genet. 2005: putative inversions between human and chimp genomes (23/27 exp. validated) - Three of those were polymorphic in 10 european individuals Levy et al., PLOS Biol. 2007: - 90 inversions identified by direct comparison of the C. Venter and the HG18 reference genome sequence assemblies Korbel et al., Science 2007: - ~3 kb fragments pair-end mapping in 2 individuals of european and african ancestry inversion brkpts. by genomic comparison with human ref. sequence Tuzun et al., Nat. Genet. 2005; Kidd et al., Nature 2008: - Fosmid pair-end mapping in 9 individuals of different geographic origins inversions validated by FISH, fingerprint analysis or breakpoint sequencing And...: - Several individual genome sequences, 1000 Genomes Project, etc. Current inversion data Total of 850 inversions in the Database of Genomic Variants (but many redundant!) Chromosomal distribution Size distribution Inversions CNVs Frequency of inversions Obs. Dist. Exp. Dist chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chr20 chr21 chr22 chrx chry 12

13 INVFEST project (INVersion Functional & Evolutionary Studies) Large-scale genomic information Latest data on human predicted inversions Traditional studies on inversions Functional and evolutionary impact of inversions Objectives and main questions Structural variation data 1 Catalogue of human inversions Human genome sequences HapMap data 2 Evolutionary history of inversions Effect on nucleotide variation 4 Other species genomes Functional annotation Functional consequences of inversions. 3 Gene-expression levels 13

14 Main scientific contributions Complete characterization of human inversions. Biological consequences of structural variants. Determine inversion genetic effects and adaptive value. Evaluate/Develop tools for study of inversions. Catalogue of human inversions Combination of predictions from different studies resulted in 364 independent inversions in humans (354 new): 49 Levy et al (90 indep. inv.) 56 6 (1) (1) 203 (3) Korbel et al (91 indep. inv.) Kidd et al (245 indep. inv.) (Martínez and Cáceres, unpub. data) 14

15 Inversions in other species Drosophila Inversions have been studied for more than 80 years and thousands of inversions have been described as fixed Or polymorphic differences Few inversions are known in other organisms: Mechanisms of generation of SVs SVs are typically generated during DNA break-induced repair, recombination or replication by different possible mechanisms: Non-allelic homologous recombination (NAHR) (duplications, deletions, inversions, translocations ) Non-homologous end joining (NHEJ) and microhomology mediated end joining (MMEJ) (deletions, inversions?) Transposition of transposable elements (insertions, deletions) Fork Stalling and Template Switching (FOsTEs) 15

16 Non-allelic homologous recombination Intra or interchomosomal recombination between copies of a sequence in different genomic positions: Non-homologous end joining Mechanism of repair of double-strand breaks that typically utilizes short homologous DNA sequences (microhomology) to guide repair: 16

17 SV breakpoints examples Inferred mechanisms of SVs Kidd et al., Nature 2008 (Fosmid PEM) Lam et al., Nature Biotech (different methods) 17

18 Biological relevance of SVs Currently identified structural variants comprise many more bases of the human genome than SNPs and could have a much bigger biological impact Functional effects of SVs Altered gene dosage and expression (CNVs) Disruption of gene or regulatory elements (insertion, deletions, inversions) Gene fusion (deletions, inversions) Change in the exon-intron structure (insertion, deletions, CNVs, inversions) Modification of gene regulatory regions (insertion, deletions, CNVs, inversions) Indirect effects though increased susceptibility of genomic rearrangements (CNVs, inversions) 18

19 Functional consequences of SVs Genomic location of SVs Distribution of SVs tends to be biased against genes and other functional elements Functional impact of CNVs by type, frequency and population (Conrad et al., Nature 2010) 19

20 Genomic location of SVs Distribution of breakpoints of 114 precisely located inversions Frequency of inversions % (76 inv.) 22.8% (26 inv.) 12 inversions (10.5%) (7 inv.) (5 inv.) Intergenic Intronic 1 mrna 2 mrna (Martínez and Cáceres, unpub. data) Functional categories enriched in CNVs 20

21 Functional categories enriched in CNVs Cell adhesion Sensory perception Synapse organization Synaptic transmission Nervous system development Defense response Immune response CNVs and complex diseases 21

22 Inversions and genomic disorders CNV example #1: CCL3L1 CCL3L1: chemokine (C-C motif) ligand 3-like 1 (González et al., Science. 2005) 22

23 CNV example #2: Amylase gene (Perry et al., Nat. Genet. 2007) Japanese ind. high starch (14 copies) Biaka ind. low starch (6 copies) Chimpanzee low starch (2 copies) Inversion example #1: Chr17q inversion (Stefansson et al., Nat. Genet. 2005) Inversion originated through NAHR between kb SD blocks Found mainly in Europan populations at ~20% frequency Possibly positively selected through increased fertility of carrier females 23

24 Inversion example #2: ChrX inversion FLNA EMD EMD FLNA 37.4 Kb (Small et al., Nat. Genet. 1997) Inversion example #2: ChrX inversion 24