A method or system for identification of a causative mutation causing a phenotype of interest in a test sample

A test sample, purpose technology, applied in the field of identification of disease-causing mutations or systems that lead to the phenotype of interest in the test sample, can solve the problems of costing money, requiring time, limiting pipeline throughput, etc.

Active Publication Date: 2021-04-09
OXFORD UNIV INNOVATION LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]Secondly, the need for outcrossing represents a quantitative limitation on typical mutation discovery pipelines, since outcrossing needs to go through at least (and often more than) one reproductive cycle, which requires time and costs money, thus limiting the throughput of the pipeline

Method used

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  • A method or system for identification of a causative mutation causing a phenotype of interest in a test sample
  • A method or system for identification of a causative mutation causing a phenotype of interest in a test sample
  • A method or system for identification of a causative mutation causing a phenotype of interest in a test sample

Examples

Experimental program
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example 1

[0530] Example 1: Mutations found in the gene for the plant-enhancing protein RHO GTPASES that impair fertility (Case B)

[0531] Several independent mutant lines were generated by irradiating N. polymorpha with UV-B. The mutant lines were divided into two phenotypic groups: some with straight rhizoids ( Figure 8 A) and intact epidermis ( Figure 9 A), some with wavy rhizoids ( Figure 8 B) and stretched epidermis ( Figure 9 B).

[0532] We aimed to identify causative mutations in UV4.32 mutant lines with wavy rhizoids and elongated cuticles. DNA was extracted from UV4.32 mutants with wavy rhizoids and elongated cuticles using whole plants as samples and standard DNA phenol-chloroform-IAA. The genome of UV4.32 and seven independent mutant lines with straight rhizoids and intact epidermis were sequenced using Illumina's HiSeq-2000 platform technology.

[0533]Raw reads were fine-tuned for quality using Trimmomatic-0.32 and normalized using Khmer0.7.1 with a k-mer size o...

example 2

[0542] Example 2: A mutation leading to resistance to chlorsulfuron was found in the acetolactate synthase gene (case A)

[0543] Polymorpha polymorpha was irradiated with UV-B and seven independent mutant lines resistant to the herbicide chlorsulfuron-methyl were identified. Chlorsulfuron resistance was determined from surviving Diana polymorpha plants after two weeks of exposure to a lethal dose of chlorsulfuron (0.1 ppm dose, a dose sufficient to kill 100% of wild-type plants).

[0544] Since all mutant plants had the same phenotype of chlorsulfuron resistance, we assumed that they each contained the same pathogenic mutation. The chlorsulfuron-resistant mutants were compared to a reference genome of over 100,000 mismatches identified individually, and we first filtered for mismatches that were also present in the M0 wild-type genome ( Figure 11 , the 2 leftmost scatter boxes).

[0545] To test the efficiency of the allelism-based version of the pipeline, we applied it to...

example 3

[0550] Example 3: A mutation leading to resistance to chlorsulfuron was found in the acetolactate synthase gene (AB case)

[0551] To increase the power of the pipelines exemplified in Examples 1 and 2, we combined two approaches: In this example of the pipeline, the Find the causative mutation in the mismatch group that exists.

[0552] Using 3 chlorsulfuron-sensitive mutagenized lines, we filtered 4 of 11 chlorsulfuron-resistant-specific mismatches previously identified as consistent with the expected mutation signature and in the coding sequence of the gene , which ended up leaving us with only 4 candidate mutations (Table 4) that were predicted to result in changes in the amino acid sequence of the protein.

[0553] This represents a 20% to 30% increase in pipeline capacity compared to the pipeline exemplified in Example 2 alone. Because the capacity of the pipelines in Examples 1 and 2 increases with the number of allelic and non-allelic minus lines, respectively, we pr...

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Abstract

A method for identifying a mutation associated with a phenotype of interest in a non- vascular plant, wherein the method comprises (a) aligning the DNA sequence of a reference DNA sequence and identifying a first set of sequence mismatches between the two sequences; wherein the test sample is from a mutagenized non-vascular plant; (b) aligning the DNA sequence of at least one comparison sample to the reference DNA sequence and identifying a second set of sequence mismatches between the two sequences; (c) filtering the first set of mismatches with respect to the second set of mismatches to identify a subset of mismatches that are unique to the first set of mismatches, wherein the subset of mismatches are candidate mutations for the causative mutation; wherein the test sample is from a non-vascular plant exhibiting the phenotype of interest and wherein the at least one comparison sample is from an independent non- vascular plant of the same genus that does not exhibit the phenotype of interest; and wherein the reference DNA sequence is a known reference sequence for a non-vascular plant of the genus. In addition, a method for identifying a mutation associated with a phenotype of interest in a non-vascular plant, wherein the method comprises a) aligning the DNA sequence of a reference DNA sequence and identifying a first set of sequence mismatches between the two sequences; wherein the test sample is from a mutagenized non-vascular plant; (b) aligning the DNA sequence of at least one comparison sample to the reference DNA sequence and identifying a second set of sequence mismatches between the two sequences; (c) filtering the first set of mismatches with respect to the second set of sequence mismatches to identify a subset of mismatches that are common to the first and second sets of sequence mismatches wherein the test sample and the comparison sample(s) are from independent non-vascular plants exhibiting the phenotype of interest and wherein the independent non-vascular plants are the same genus; and wherein the reference DNA sequence is a known reference sequence or a non-vascular plant of the genus or a non-vascular plant of the genus.

Description

technical field [0001] The present invention generally relates to methods or systems for identifying pathogenic mutations that cause a phenotype of interest in a test sample. Background technique [0002] Identifying genotypes associated with phenotypes of interest is critical in many applications of plant biology. Mutation discovery following mutagenesis experiments typically involves outcrossing mutants with wild-type plants, generating large populations of wild-type and mutants, and identifying mutations that occur only in large numbers of mutants. Doing so allows genome recombination of wild-type and mutants, thus reducing the number of background mutations in the mutant genome and increasing the chances of identifying disease-causing mutations. However, this preliminary step comes at a cost. [0003] First, the need for outcrossing represents a qualitative limitation on typical mutation discovery pipelines, since sterile mutants cannot interbreed. Many mutations (whe...

Claims

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Application Information

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IPC IPC(8): A01H1/04C12N15/00G16B20/20G16B20/50G16B30/10
CPCG16B20/20G16B30/10G16B20/50
Inventor C·钱皮恩L·多兰
Owner OXFORD UNIV INNOVATION LTD
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