Rapid High Resolution Single Nucleotide Polymorphism–Comparative Genome Hybridization Mapping in <i>Caenorhabditis elegans</i>

Flibotte, Stéphane; Edgley, Mark L.; Maydan, Jason S.; Taylor, John; Zapf, Rick; Waterston, Robert H.; Moerman, Donald G.

doi:10.1534/genetics.108.096487

Cited by 24 publications

(33 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This renormalization is necessary to account for local bias, which varies both among and within experiments and makes the detection of SNPs more difficult since artifacts associated with a strong local bias in the log 2 ratio could easily be confused with the signature expected for a SNP. Unlike previous observations that mutations near the centers of 25-mer probes are most inhibitory to efficient hybridization (Sharp et al 2007), we observed that mutations located away from the glass slide and freely floating in the solution closer to the 59-ends of our 50-mer probes produced a larger perturbation to the hybridization process, with a maximum perturbation at 7 bases in from the 59-end (probably due to steric effects; again see Figure 1 in Flibotte et al 2009). The location of the Multiple adjacent overlapping probes targeted the point mutation, so its effect on hybridization was assayed several times.…”

contrasting

confidence: 98%

“…In a previous study (Flibotte et al 1 2009) we have shown that a window of $20 bases contains a strong log 2 ratio signal (see Figure 1 in Flibotte et al 2009), and since we require about four probes to target the mutated site, this allows a maximum probe spacing of $5 bases. The plot in Flibotte's figure also shows that it would be useful to target both strands and use the small shift in the peak position on opposite strands to help distinguish single nucleotide polymorphisms (SNPs) from artifacts.…”

mentioning

confidence: 95%

See 1 more Smart Citation

De Novo Identification of Single Nucleotide Mutations in Caenorhabditis elegans Using Array Comparative Genomic Hybridization

et al. 2009

Self Cite

View full text Add to dashboard Cite

Array comparative genomic hybridization (aCGH) has been used primarily to detect copy-number variants between two genomes. Here we report using aCGH to detect single nucleotide mutations on oligonucleotide microarrays with overlapping 50-mer probes. This technique represents a powerful method for rapidly detecting novel homozygous single nucleotide mutations in any organism with a sequenced reference genome.

show abstract

contrasting

confidence: 98%

mentioning

confidence: 95%

De Novo Identification of Single Nucleotide Mutations in Caenorhabditis elegans Using Array Comparative Genomic Hybridization

et al. 2009

Self Cite

View full text Add to dashboard Cite

show abstract

“…The color key identifying the type of mutation is provided in the inset, and the first letter represents the reference nucleotide while the second letter represents the mutated nucleotide. available where traits can be mapped to a 5-map-unit interval, or even a smaller interval, after a single cross (Michelmore et al 1991;Jakubowski and Kornfeld 1999;Wicks et al 2001;Swan et al 2002;Davis et al 2005;Flibotte et al 2009). In combination with deep sequencing these methods will provide an unprecedented resource for new forward genetics discoveries.…”

Section: Discussionmentioning

confidence: 99%

Whole-Genome Profiling of Mutagenesis inCaenorhabditis elegans

et al. 2010

Self Cite

View full text Add to dashboard Cite

Deep sequencing offers an unprecedented view of an organism's genome. We describe the spectrum of mutations induced by three commonly used mutagens: ethyl methanesulfonate (EMS), N-ethyl-Nnitrosourea (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.

show abstract

“…Each SNP was represented on the microarray by an average of almost 40 50-mer oligonucleotides targeting both the AF16 (cb3 assembly) and the HK104 sequences. The typical spacing between adjacent oligonucleotides was 2 bases and the targeted strand was selected to ensure the SNPs were as far as possible from the slide to maximize the SNP signal (Flibotte et al 2009). The current microarray design is available to the community under the name ''080211_Moerman_cbriggsae_CGH'' and researchers are encouraged to contact the authors to inquire about potential future releases.…”

Section: Methodsmentioning

confidence: 99%

New Tools for Investigating the Comparative Biology of Caenorhabditis briggsae and C. elegans

et al. 2010

Self Cite

View full text Add to dashboard Cite

Comparative studies of Caenorhabditis briggsae and C. elegans have provided insights into gene function and developmental control in both organisms. C. elegans is a well developed model organism with a variety of molecular and genetic tools to study gene functions. In contrast, there are only very limited tools available for its closest relative, C. briggsae. To take advantage of the full potential of this comparative approach, we have developed several genetic and molecular tools to facilitate functional analysis in C. briggsae. First, we designed and implemented an SNP-based oligonucleotide microarray for rapid mapping of genetic mutants in C. briggsae. Second, we generated a mutagenized frozen library to permit the isolation of targeted deletions and used the library to recover a deletion mutant of cbr-unc-119 for use as a transgenic marker. Third, we used the cbr-unc-119 mutant in ballistic transformation and generated fluorescently labeled strains that allow automated lineaging and cellular resolution expression analysis. Finally, we demonstrated the potential of automated lineaging by profiling expression of egl-5, hlh-1, and pha-4 at cellular resolution and by detailed phenotyping of the perturbations on the Wnt signaling pathway. These additions to the experimental toolkit for C. briggsae should greatly increase its utility in comparative studies with C. elegans. With the emerging sequence of nematode species more closely related to C. briggsae, these tools may open novel avenues of experimentation in C. briggsae itself. C OMPARATIVE analysis of genes between related species is a powerful experimental approach, allowing one to exploit nature's experiments to illuminate the sequence basis of function. Commonly, conservation of sequence is used to delineate functional elements in a genome and is particularly effective with protein-coding sequence. However, in many instances differences in sequence may also be informative. For example, the small size of transcription factor binding sites allows for generation of new sites and loss of old sites, masking the conservation of functions (Andolfatto 2005). In other cases, a change in sequence is associated with a change in function that sheds light on the underlying mechanisms of gene action. In these instances, simple sequence comparisons are inadequate; they need to be supplemented with functional analysis.For comparative functional analysis of Caenorhabditis elegans, C. briggsae remains the preferred species. Although it shows an estimated 1.6 substitutions per neutral site with C. elegans (Stein et al. 2003), it remains the closest identified living relative. Both are selffertilizing hermaphrodites and have very similar morphology. Recent evidence suggests that the developmental cell lineage is remarkably constant between the two species (Zhao et al. 2008). However, changes in cellular signaling underlying these patterns have been revealed by quantitative study of development after cell ablation or manipulation of the signaling molecules (Felix 2007)...

show abstract

Rapid High Resolution Single Nucleotide Polymorphism–Comparative Genome Hybridization Mapping in Caenorhabditis elegans

Cited by 24 publications

References 14 publications

De Novo Identification of Single Nucleotide Mutations in Caenorhabditis elegans Using Array Comparative Genomic Hybridization

De Novo Identification of Single Nucleotide Mutations in Caenorhabditis elegans Using Array Comparative Genomic Hybridization

Whole-Genome Profiling of Mutagenesis inCaenorhabditis elegans

New Tools for Investigating the Comparative Biology of Caenorhabditis briggsae and C. elegans

Contact Info

Product

Resources

About