Stephane Flibotte

Oligonucleotide Microarray Analysis of Genomic Imbalance in Children with Mental Retardation

The cause of mental retardation in one-third to one-half of all affected individuals is unknown. Microscopically detectable chromosomal abnormalities are the most frequently recognized cause, but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause. Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants. We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis, by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays. We found de novo deletions as small as 178 kb in eight cases, de novo duplications as small as 1.1 Mb in two cases, and unsuspected mosaic trisomy 9 in another case. This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation.

The million mutation project: A new approach to genetics in Caenorhabditis elegans

We have created a library of 2007 mutagenized Caenorhabditis elegans strains, each sequenced to a target depth of 15-fold coverage, to provide the research community with mutant alleles for each of the worms more than 20,000 genes. The library contains over 800,000 unique single nucleotide variants (SNVs) with an average of eight nonsynonymous changes per gene and more than 16,000 insertion/deletion (indel) and copy number changes, providing an unprecedented genetic resource for this multicellular organism. To supplement this collection, we also sequenced 40 wild isolates, identifying more than 630,000 unique SNVs and 220,000 indels. Comparison of the two sets demonstrates that the mutant collection has a much richer array of both nonsense and missense mutations than the wild isolate set. We also find a wide range of rDNA and telomere repeat copy number in both sets. Scanning the mutant collection for molecular phenotypes reveals a nonsense suppressor as well as strains with higher levels of indels that harbor mutations in DNA repair genes and strains with abundant males associated with him mutations. All the strains are available through the Caenorhabditis Genetics Center and all the sequence changes have been deposited in WormBase and are available through an interactive website.

Whole-Genome Profiling of Mutagenesis in Caenorhabditis elegans

Deep sequencing offers an unprecedented view of an organisms genome. We describe the spectrum of mutations induced by three commonly used mutagens: ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), and ultraviolet trimethylpsoralen (UV/TMP) in the nematode Caenorhabditis elegans. Our analysis confirms the strong GC to AT transition bias of EMS. We found that ENU mainly produces A to T and T to A transversions, but also all possible transitions. We found no bias for any specific transition or transversion in the spectrum of UV/TMP-induced mutations. In 10 mutagenized strains we identified 2723 variants, of which 508 are expected to alter or disrupt gene function, including 21 nonsense mutations and 10 mutations predicted to affect mRNA splicing. This translates to an average of 50 informative mutations per strain. We also present evidence of genetic drift among laboratory wild-type strains derived from the Bristol N2 strain. We make several suggestions for best practice using massively parallel short read sequencing to ensure mutation detection.

Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon

We have generated a recombinant Mos1 transposon that can insert up to 45-kb transgenes into the Caenorhabditis elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (∼60% of injected animals). Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae. We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.

Impact of whole genome amplification on analysis of copy number variants

Large-scale copy number variants (CNVs) have recently been recognized to play a role in human genome variation and disease. Approaches for analysis of CNVs in small samples such as microdissected tissues can be confounded by limited amounts of material. To facilitate analyses of such samples, whole genome amplification (WGA) techniques were developed. In this study, we explored the impact of Phi29 multiple-strand displacement amplification on detection of CNVs using oligonucleotide arrays. We extracted DNA from fresh frozen lymph node samples and used this for amplification and analysis on the Affymetrix Mapping 500k SNP array platform. We demonstrated that the WGA procedure introduces hundreds of potentially confounding CNV artifacts that can obscure detection of bona fide variants. Our analysis indicates that many artifacts are reproducible, and may correlate with proximity to chromosome ends and GC content. Pair-wise comparison of amplified products considerably reduced the number of apparent artifacts and partially restored the ability to detect real CNVs. Our results suggest WGA material may be appropriate for copy number analysis when amplified samples are compared to similarly amplified samples and that only the CNVs with the greatest significance values detected by such comparisons are likely to be representative of the unamplified samples.

Widespread, focal copy number variations (CNV) and whole chromosome aneuploidies in Trypanosoma cruzi strains revealed by array comparative genomic hybridization

BackgroundTrypanosoma cruzi is a protozoan parasite and the etiologic agent of Chagas disease, an important public health problem in Latin America. T. cruzi is diploid, almost exclusively asexual, and displays an extraordinarily diverse population structure both genetically and phenotypically. Yet, to date the genotypic diversity of T. cruzi and its relationship, if any, to biological diversity have not been studied at the whole genome level.ResultsIn this study, we used whole genome oligonucleotide tiling arrays to compare gene content in biologically disparate T. cruzi strains by comparative genomic hybridization (CGH). We observed that T. cruzi strains display widespread and focal copy number variations (CNV) and a substantially greater level of diversity than can be adequately defined by the current genetic typing methods. As expected, CNV were particularly frequent in gene family-rich regions containing mucins and trans-sialidases but were also evident in core genes. Gene groups that showed little variation in copy numbers among the strains tested included those encoding protein kinases and ribosomal proteins, suggesting these loci were less permissive to CNV. Moreover, frequent variation in chromosome copy numbers were observed, and chromosome-specific CNV signatures were shared by genetically divergent T. cruzi strains.ConclusionsThe large number of CNV, over 4,000, reported here uphold at a whole genome level the long held paradigm of extraordinary genome plasticity among T. cruzi strains. Moreover, the fact that these heritable markers do not parse T. cruzi strains along the same lines as traditional typing methods is strongly suggestive of genetic exchange playing a major role in T. cruzi population structure and biology.

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

BackgroundGenomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.ResultsWe evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.ConclusionWe observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.

Copy number variation in the genomes of twelve natural isolates of Caenorhabditis elegans

BackgroundCopy number variation is an important component of genetic variation in higher eukaryotes. The extent of natural copy number variation in C. elegans is unknown outside of 2 highly divergent wild isolates and the canonical N2 Bristol strain.ResultsWe have used array comparative genomic hybridization (aCGH) to detect copy number variation in the genomes of 12 natural isolates of Caenorhabditis elegans. Deletions relative to the canonical N2 strain are more common in these isolates than duplications, and indels are enriched in multigene families on the autosome arms. Among the strains in our study, the Hawaiian and Madeiran strains (CB4856 and JU258) carry the largest number of deletions, followed by the Vancouver strain (KR314). Overall we detected 510 different deletions affecting 1136 genes, or over 5% of the genes in the canonical N2 genome. The indels we identified had a median length of 2.7 kb. Since many deletions are found in multiple isolates, deletion loci were used as markers to derive an unrooted tree to estimate genetic relatedness among the strains.ConclusionCopy number variation is extensive in C. elegans, affecting over 5% of the genes in the genome. The deletions we have detected in natural isolates of C. elegans contribute significantly to the number of deletion alleles available to researchers. The relationships between strains are complex and different regions of the genome possess different genealogies due to recombination throughout the natural history of the species, which may not be apparent in studies utilizing smaller numbers of genetic markers.

Transcription of foreign DNA in Escherichia coli

Propagation of heterologous DNA in E. coli host cells is central to molecular biology. DNA constructs are often engineered for expression of recombinant protein in E. coli, but the extent of incidental transcription arising from natural regulatory sequences in cloned DNA remains underexplored. Here, we have used programmable microarrays and RT-PCR to measure, comprehensively, the transcription of H. influenzae, P. aeruginosa, and human DNA propagating in E. coli as bacterial artificial chromosomes. We find evidence that at least half of all H. influenzae genes are transcribed in E. coli. Highly transcribed genes are principally involved in energy metabolism, and their proximal promoter regions are significantly enriched with E. coli sigma(70) (also known as RpoD) binding sites. H. influenzae genes acquired from an ancient bacteriophage Mu insertion are also highly transcribed. Compared with H. influenzae, a smaller proportion of P. aeruginosa genes are transcribed in E. coli, and in E. coli there is punctuated transcription of human DNA. The presence of foreign DNA in E. coli disturbs the host transcriptional profile, with expression of the E. coli phage shock protein operon and the flagellar gene cluster being particularly strongly up-regulated. While cross-species transcriptional activation is expected to be enabling for horizontal gene transfer in bacteria, incidental expression of toxic genes can be problematic for DNA cloning. Ongoing characterization of cross-expression will help inform the design of biosynthetic gene clusters and synthetic microbial genomes.

DNA Synthesis Generates Terminal Duplications That Seal End-to-End Chromosome Fusions

The fate of chromosomes that become catastrophically fused at their ends is revealed in the nematode worm. End-to-end chromosome fusions that occur in the context of telomerase deficiency can trigger genomic duplications. For more than 70 years, these duplications have been attributed solely to breakage-fusion-bridge cycles. To test this hypothesis, we examined end-to-end fusions isolated from Caenorhabditis elegans telomere replication mutants. Genome-level rearrangements revealed fused chromosome ends having interrupted terminal duplications accompanied by template-switching events. These features are very similar to disease-associated duplications of interstitial segments of the human genome. A model termed Fork Stalling and Template Switching has been proposed previously to explain such duplications, where promiscuous replication of large, noncontiguous segments of the genome occurs. Thus, a DNA synthesis–based process may create duplications that seal end-to-end fusions, in the absence of breakage-fusion-bridge cycles.