Dataset Information

Rapid, precise quantification of large DNA excisions and inversions by ddPCR.

ABSTRACT: The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (>?20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.

SUBMITTER: Watry HL

PROVIDER: S-EPMC7483445 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Rapid, precise quantification of large DNA excisions and inversions by ddPCR.

Watry Hannah L HL Feliciano Carissa M CM Gjoni Ketrin K Takahashi Gou G Miyaoka Yuichiro Y Conklin Bruce R BR Judge Luke M LM

Scientific reports 20200910 1

The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of exc ...[more]

PMID: 32913194

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Project description:BackgroundMass spectrometry based quantification of peptides can be performed using the iTRAQ reagent in conjunction with mass spectrometry. This technology yields information about the relative abundance of single peptides. A method for the calculation of reliable quantification information is required in order to obtain biologically relevant data at the protein expression level.ResultsA method comprising sound error estimation and statistical methods is presented that allows precise abundance analysis plus error calculation at the peptide as well as at the protein level. This yields the relevant information that is required for quantitative proteomics. Comparing the performance of our method named Quant with existing approaches the error estimation is reliable and offers information for precise bioinformatic models. Quant is shown to generate results that are consistent with those produced by ProQuant, thus validating both systems. Moreover, the results are consistent with that of Mascot 2.2. The MATLAB scripts of Quant are freely available via http://www.protein-ms.de and http://sourceforge.net/projects/protms/, each under the GNU Lesser General Public License.ConclusionThe software Quant demonstrates improvements in protein quantification using iTRAQ. Precise quantification data can be obtained at the protein level when using error propagation and adequate visualization. Quant integrates both and additionally provides the possibility to obtain more reliable results by calculation of wise quality measures. Peak area integration has been replaced by sum of intensities, yielding more reliable quantification results. Additionally, Quant allows the combination of quantitative information obtained by iTRAQ with peptide and protein identifications from popular tandem MS identification tools. Hence Quant is a useful tool for the proteomics community and may help improving analysis of proteomic experimental data. In addition, we have shown that a lognormal distribution fits the data of mass spectrometry based relative peptide quantification.

Dataset Information

Rapid, precise quantification of large DNA excisions and inversions by ddPCR.

Publications

Rapid, precise quantification of large DNA excisions and inversions by ddPCR.

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