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A massively parallel pipeline to clone DNA variants and examine molecular phenotypes of human disease mutations.

Wei X, Das J, Fragoza R, Liang J, Bastos de Oliveira FM, Lee HR, Wang X, Mort M, Stenson PD, Cooper DN, Lipkin SM, Smolka MB, Yu H - PLoS Genet. (2014)

Bottom Line: We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles.We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions.The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Weill Cornell College of Medicine, New York, New York, United States of America; Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

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Identifying usable clones from Clone-seq.(a) Schematic illustrating criteria used to determine which of the clones generated by our Clone-seq pipeline are usable for further assays – green ticks indicate usable clones, while red crosses indicate clones that cannot be used. (b) Variation of S across different mutagenesis attempts that either contain or do not contain the desired mutation as confirmed by Sanger sequencing.
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pgen-1004819-g002: Identifying usable clones from Clone-seq.(a) Schematic illustrating criteria used to determine which of the clones generated by our Clone-seq pipeline are usable for further assays – green ticks indicate usable clones, while red crosses indicate clones that cannot be used. (b) Variation of S across different mutagenesis attempts that either contain or do not contain the desired mutation as confirmed by Sanger sequencing.

Mentions: The first step of our pipeline is a massively parallel approach, termed Clone-seq, designed to leverage the power of next-generation sequencing to generate a large number of mutant alleles using site-directed mutagenesis in a rapid and cost-effective manner. Current protocols for site-directed mutagenesis require picking individual colonies and sequencing each colony using Sanger sequencing to identify the correct clone [15]. This standard approach is both labor-intensive and expensive; therefore, it does not scale up to genome-wide surveys. In Clone-seq, we put one colony of each mutagenesis attempt into one pool (Fig. 1a; in other words, each pool contains one and only one colony for each desired mutation) and combine multiple pools through multiplexing for one Illumina sequencing run [16]. Colonies for generating different mutations of the same gene can be put into the same pool, which can be easily distinguished computationally when processing the sequencing results. This is true even for mutations occurring at the same site (Fig. 2a, Text S1).


A massively parallel pipeline to clone DNA variants and examine molecular phenotypes of human disease mutations.

Wei X, Das J, Fragoza R, Liang J, Bastos de Oliveira FM, Lee HR, Wang X, Mort M, Stenson PD, Cooper DN, Lipkin SM, Smolka MB, Yu H - PLoS Genet. (2014)

Identifying usable clones from Clone-seq.(a) Schematic illustrating criteria used to determine which of the clones generated by our Clone-seq pipeline are usable for further assays – green ticks indicate usable clones, while red crosses indicate clones that cannot be used. (b) Variation of S across different mutagenesis attempts that either contain or do not contain the desired mutation as confirmed by Sanger sequencing.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263371&req=5

pgen-1004819-g002: Identifying usable clones from Clone-seq.(a) Schematic illustrating criteria used to determine which of the clones generated by our Clone-seq pipeline are usable for further assays – green ticks indicate usable clones, while red crosses indicate clones that cannot be used. (b) Variation of S across different mutagenesis attempts that either contain or do not contain the desired mutation as confirmed by Sanger sequencing.
Mentions: The first step of our pipeline is a massively parallel approach, termed Clone-seq, designed to leverage the power of next-generation sequencing to generate a large number of mutant alleles using site-directed mutagenesis in a rapid and cost-effective manner. Current protocols for site-directed mutagenesis require picking individual colonies and sequencing each colony using Sanger sequencing to identify the correct clone [15]. This standard approach is both labor-intensive and expensive; therefore, it does not scale up to genome-wide surveys. In Clone-seq, we put one colony of each mutagenesis attempt into one pool (Fig. 1a; in other words, each pool contains one and only one colony for each desired mutation) and combine multiple pools through multiplexing for one Illumina sequencing run [16]. Colonies for generating different mutations of the same gene can be put into the same pool, which can be easily distinguished computationally when processing the sequencing results. This is true even for mutations occurring at the same site (Fig. 2a, Text S1).

Bottom Line: We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles.We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions.The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Weill Cornell College of Medicine, New York, New York, United States of America; Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

Show MeSH