A Rapid, SuperSelective Method for Detection of Single Nucleotide Variants in Caenorhabditis elegans

With the widespread use of single nucleotide variants generated through mutagenesis screens and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end-point PCR in Caenorhabditis elegans. Each SS primer consists of a 5′ “anchor” that hybridizes to the template, followed by a noncomplementary “bridge,” and a “foot” corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3′ nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length and sequence composition of each SS primer segment affected selectivity and efficiency in various genetic contexts in order to develop simple rules for primer design that allow for differentiation between alleles over a broad range of annealing temperatures. Manipulating bridge length affected amplification efficiency, while modifying the foot sequence altered discriminatory power. Changing the anchor position enabled SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. After defining primer design parameters, we demonstrated the utility of SS primers for genotyping crude C. elegans lysates, suggesting that this approach could also be used for SNP mapping and screening of CRISPR mutants. Further, since SS primers reliably detect point mutations, this method has potential for broad application in all genetic systems.

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A rapid, super-selective method for detection of single nucleotide variants in C. elegans

10.1101/2020.04.01.020818 ◽

2020 ◽

Author(s):

Denis Touroutine ◽

Jessica E. Tanis

Keyword(s):

Low Cost ◽

Primer Design ◽

Site Directed Mutagenesis ◽

Amplification Efficiency ◽

Single Nucleotide Variants ◽

Nucleotide Substitutions ◽

Single Nucleotide ◽

Colony Pcr ◽

Sequence Composition ◽

C Elegans

ABSTRACTWith the widespread use of single nucleotide variants generated through mutagenesis screens, the million mutation project, and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end point PCR in Caenorhabditis elegans. Each SS primer consists of a 5’ “anchor” that hybridizes to the template, followed by a non-complementary “bridge,” and a “foot” corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3’ nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length, stability, and sequence composition of each SS primer segment affected selectivity and efficiency in order to develop simple rules for primer design that allow for distinction between any mismatches in various genetic contexts over a broad range of annealing temperatures. Manipulating bridge length affects amplification efficiency, while modifying the foot sequence can increase discriminatory power. Flexibility in the positioning of the anchor enables SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. In summary, we have demonstrated flexibility in design of SS primers and their utility for genotyping in C. elegans. Since SS primers reliably detect single nucleotide variants, we propose that this method could have broad application for SNP mapping, screening of CRISPR mutants, and colony PCR to identify successful site-directed mutagenesis constructs.

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A genotyping method combining primer competition PCR with HRM analysis to identify point mutations in Duchenne animal models

Scientific Reports ◽

10.1038/s41598-020-74173-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Haizpea Lasa-Fernandez ◽

Laura Mosqueira-Martín ◽

Ainhoa Alzualde ◽

Jaione Lasa-Elgarresta ◽

Ainara Vallejo-Illarramendi

Keyword(s):

High Throughput Screening ◽

Point Mutations ◽

Mdx Mouse ◽

Single Nucleotide Variants ◽

High Resolution Melt ◽

Nucleotide Substitutions ◽

Special Equipment ◽

Single Nucleotide ◽

Screening Experiments ◽

Genotyping Method

Abstract Dystrophin-null sapje zebrafish is an excellent model for better understanding the pathological mechanisms underlying Duchenne muscular dystrophy, and it has recently arisen as a powerful tool for high-throughput screening of therapeutic candidates for this disease. While dystrophic phenotype in sapje larvae can be easily detected by birefringence, zebrafish genotyping is necessary for drug screening experiments, where the potential rescue of larvae phenotype is the primary outcome. Genotyping is also desirable during colony husbandry since heterozygous progenitors need to be selected. Currently, sapje zebrafish are genotyped through techniques involving sequencing or multi-step PCR, which are often costly, tedious, or require special equipment. Here we report a simple, precise, cost-effective, and versatile PCR genotyping method based on primer competition. Genotypes can be resolved by standard agarose gel electrophoresis and high-resolution melt assay, the latter being especially useful for genotyping a large number of samples. Our approach has shown high sensitivity, specificity, and reproducibility in detecting the A/T point mutation in sapje zebrafish and the C/T mutation in the mdx mouse model of Duchenne. Hence, this method can be applied to other single nucleotide substitutions and may be further optimized to detect small insertions and deletions. Given its robust performance with crude DNA extracts, our strategy may be particularly well-suited for detecting single nucleotide variants in poor-quality samples such as ancient DNA or DNA from formalin-fixed, paraffin-embedded material.

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Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

10.1101/2021.09.14.460298 ◽

2021 ◽

Author(s):

Jeffrey C Medley ◽

Shilpa Hebbar ◽

Joel T Sydzyik ◽

Anna Y. Zinovyeva

Keyword(s):

Caenorhabditis Elegans ◽

Genome Editing ◽

Dna Breaks ◽

Nucleotide Substitutions ◽

Paternal Genome ◽

Single Nucleotide ◽

C Elegans ◽

Homology Directed Repair ◽

Maternal Genome ◽

Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

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Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos

Communications Biology ◽

10.1038/s42003-019-0745-3 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 15

Author(s):

Hye Kyung Lee ◽

Harold E. Smith ◽

Chengyu Liu ◽

Michaela Willi ◽

Lothar Hennighausen

Keyword(s):

Point Mutations ◽

Mouse Embryos ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Base Editing ◽

Genome Wide ◽

Wide Range ◽

Family Based ◽

Correct Point ◽

Adenine Base

AbstractDeaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 and ABE. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls. Therefore, an optimization of cytosine base editors is required to improve its fidelity. While the remarkable fidelity of ABE has implications for a wide range of applications, the occurrence of rare aberrant C-to-T conversions at specific target sites needs to be addressed.

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Polymorphisms in the goat β-lactoglobulin gene

Journal of Dairy Research ◽

10.1017/s0022029905000981 ◽

2005 ◽

Vol 72 (3) ◽

pp. 379-384 ◽

Cited By ~ 12

Author(s):

Maria Ballester ◽

Armand Sánchez ◽

Josep M Folch

Keyword(s):

Genetic Variants ◽

Promoter Region ◽

Point Mutations ◽

Proximal Promoter ◽

Coding Region ◽

Nucleotide Substitutions ◽

Single Nucleotide ◽

Β Lactoglobulin ◽

Pyrosequencing Technology ◽

Frequent Haplotype

β-lactoglobulin polymorphisms have been reported in the milk of different goat breeds, although no genetic variants affecting the protein have been characterized. In the present study, we amplified and sequenced the proximal promoter and the first six exons containing the entire coding region for the β-lactoglobulin gene in eleven goat breeds from Spain, France, Italy, Switzerland, Senegal and Asia to identify genetic variants. Fifteen polymorphisms were detected, nine in the promoter region and six in the exons of the β-lactoglobulin gene. All polymorphisms were single nucleotide substitutions with the exception of one deletion/insertion in the promoter region. The polymorphisms in the coding region did not produce any amino acid change. In addition, pyrosequencing technology was used to genotype four polymorphisms in the promoter region in 200 goats belonging to eleven breeds. Differences in allelic frequencies for these polymorphisms between breeds are described and a specific polymorphism for the Italian populations was identified. Finally, the analysis of association between these four promoter point mutations was investigated resulting in five haplotypes, GCGC being the most frequent haplotype in all breeds analysed.

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Computational Analysis of Genetic Code Variations Optimized for the Robustness against Point Mutations with Wobble-like Effects

Life ◽

10.3390/life11121338 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1338

Author(s):

Elena Fimmel ◽

Markus Gumbel ◽

Martin Starman ◽

Lutz Strüngmann

Keyword(s):

Genetic Code ◽

Computational Analysis ◽

Point Mutations ◽

Weighted Graph ◽

Single Nucleotide Variants ◽

Negative Effects ◽

Standard Genetic Code ◽

Single Nucleotide ◽

Random Code ◽

Optimal Weights

It is believed that the codon–amino acid assignments of the standard genetic code (SGC) help to minimize the negative effects caused by point mutations. All possible point mutations of the genetic code can be represented as a weighted graph with weights that correspond to the probabilities of these mutations. The robustness of a code against point mutations can be described then by means of the so-called conductance measure. This paper quantifies the wobble effect, which was investigated previously by applying the weighted graph approach, and seeks optimal weights using an evolutionary optimization algorithm to maximize the code’s robustness. One result of our study is that the robustness of the genetic code is least influenced by mutations in the third position—like with the wobble effect. Moreover, the results clearly demonstrate that point mutations in the first, and even more importantly, in the second base of a codon have a very large influence on the robustness of the genetic code. These results were compared to single nucleotide variants (SNV) in coding sequences which support our findings. Additionally, it was analyzed which structure of a genetic code evolves from random code tables when the robustness is maximized. Our calculations show that the resulting code tables are very close to the standard genetic code. In conclusion, the results illustrate that the robustness against point mutations seems to be an important factor in the evolution of the standard genetic code.

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Comprehensive Custom NGS Panel Validation for the Improvement of the Stratification of B-Acute Lymphoblastic Leukemia Patients

Journal of Personalized Medicine ◽

10.3390/jpm10030137 ◽

2020 ◽

Vol 10 (3) ◽

pp. 137

Author(s):

Adrián Montaño ◽

Jesús Hernández-Sánchez ◽

Maribel Forero-Castro ◽

María Matorra-Miguel ◽

Eva Lumbreras ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Wide Spectrum ◽

Lymphoblastic Leukemia ◽

Point Mutations ◽

Genetic Alterations ◽

Copy Number Variations ◽

Single Step ◽

Fusion Genes ◽

Single Nucleotide Variants ◽

Single Nucleotide

Background: B-acute lymphoblastic leukemia (B-ALL) is a hematological neoplasm of the stem lymphoid cell of the B lineage, characterized by the presence of genetic alterations closely related to the course of the disease. The number of alterations identified in these patients grows as studies of the disease progress, but in clinical practice, the conventional techniques frequently used are only capable of detecting the most common alterations. However, techniques, such as next-generation sequencing (NGS), are being implemented to detect a wide spectrum of new alterations that also include point mutations. Methods: In this study, we designed and validated a comprehensive custom NGS panel to detect the main genetic alterations present in the disease in a single step. For this purpose, 75 B-ALL diagnosis samples from patients previously characterized by standard-of-care diagnostic techniques were sequenced. Results: The use of the custom NGS panel allowed the correct detection of the main genetic alterations present in B-ALL patients, including the presence of an aneuploid clone in 14 of the samples and some of the recurrent fusion genes in 35 of the samples. The panel was also able to successfully detect a number of secondary alterations, such as single nucleotide variants (SNVs) and copy number variations (CNVs) in 66 and 46 of the samples analyzed, respectively, allowing for further refinement of the stratification of patients. The custom NGS panel could also detect alterations with a high level of sensitivity and reproducibility when the findings obtained by NGS were compared with those obtained from other conventional techniques. Conclusions: The use of this custom NGS panel allows us to quickly and efficiently detect the main genetic alterations present in B-ALL patients in a single assay (SNVs and insertions/deletions (INDELs), recurrent fusion genes, CNVs, aneuploidies, and single nucleotide polymorphisms (SNPs) associated with pharmacogenetics). The application of this panel would thus allow us to speed up and simplify the molecular diagnosis of patients, helping patient stratification and management.

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Next-generation forward genetic screens: using simulated data to improve the design of mapping-by-sequencing experiments in Arabidopsis

Nucleic Acids Research ◽

10.1093/nar/gkz806 ◽

2019 ◽

Vol 47 (21) ◽

pp. e140-e140

Author(s):

David Wilson-Sánchez ◽

Samuel Daniel Lup ◽

Raquel Sarmiento-Mañús ◽

María Rosa Ponce ◽

José Luis Micol

Keyword(s):

Point Mutations ◽

Sequencing Depth ◽

Nucleotide Polymorphisms ◽

Next Generation ◽

Genetic Screens ◽

Induced Mutations ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Forward Genetic Screens ◽

Mapping By Sequencing

Abstract Forward genetic screens have successfully identified many genes and continue to be powerful tools for dissecting biological processes in Arabidopsis and other model species. Next-generation sequencing technologies have revolutionized the time-consuming process of identifying the mutations that cause a phenotype of interest. However, due to the cost of such mapping-by-sequencing experiments, special attention should be paid to experimental design and technical decisions so that the read data allows to map the desired mutation. Here, we simulated different mapping-by-sequencing scenarios. We first evaluated which short-read technology was best suited for analyzing gene-rich genomic regions in Arabidopsis and determined the minimum sequencing depth required to confidently call single nucleotide variants. We also designed ways to discriminate mutagenesis-induced mutations from background Single Nucleotide Polymorphisms in mutants isolated in Arabidopsis non-reference lines. In addition, we simulated bulked segregant mapping populations for identifying point mutations and monitored how the size of the mapping population and the sequencing depth affect mapping precision. Finally, we provide the computational basis of a protocol that we already used to map T-DNA insertions with paired-end Illumina-like reads, using very low sequencing depths and pooling several mutants together; this approach can also be used with single-end reads as well as to map any other insertional mutagen. All these simulations proved useful for designing experiments that allowed us to map several mutations in Arabidopsis.

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Reproducibility of SNV-calling in multiple sequencing runs from single tumors

PeerJ ◽

10.7717/peerj.1508 ◽

2016 ◽

Vol 4 ◽

pp. e1508 ◽

Cited By ~ 3

Author(s):

Dakota Z. Derryberry ◽

Matthew C. Cowperthwaite ◽

Claus O. Wilke

Keyword(s):

Glioblastoma Multiforme ◽

Large Fraction ◽

Point Mutations ◽

Cancer Genome ◽

The Cancer Genome Atlas ◽

Single Nucleotide Variants ◽

Specific Point ◽

Single Nucleotide ◽

Cancer Genome Atlas ◽

Genome Atlas

We examined 55 technical sequencing replicates of Glioblastoma multiforme (GBM) tumors from The Cancer Genome Atlas (TCGA) to ascertain the degree of repeatability in calling single-nucleotide variants (SNVs). We used the same mutation-calling pipeline on all pairs of samples, and we measured the extent of the overlap between two replicates; that is, how many specific point mutations were found in both replicates. We further tested whether additional filtering increased or decreased the size of the overlap. We found that about half of the putative mutations identified in one sequencing run of a given sample were also identified in the second, and that this percentage remained steady throughout orders of magnitude of variation in the total number of mutations identified (from 23 to 10,966). We further found that using filtering after SNV-calling removed the overlap completely. We concluded that there is variation in the frequency of mutations in GBMs, and that while some filtering approaches preferentially removed putative mutations found in only one replicate, others removed a large fraction of putative mutations found in both.

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Comparative genomic analysis of the principal Cryptosporidium species that infect humans

PeerJ ◽

10.7717/peerj.10478 ◽

2020 ◽

Vol 8 ◽

pp. e10478

Author(s):

Laura M. Arias-Agudelo ◽

Gisela Garcia-Montoya ◽

Felipe Cabarcas ◽

Ana L. Galvan-Diaz ◽

Juan F. Alzate

Keyword(s):

Genomic Analysis ◽

Comparative Genomic Analysis ◽

Comparative Genomic ◽

Single Nucleotide Variants ◽

Nucleotide Substitutions ◽

Genomic Comparison ◽

Single Nucleotide ◽

Associated Proteins ◽

Species Specific ◽

Ngs Data

Cryptosporidium parasites are ubiquitous and can infect a broad range of vertebrates and are considered the most frequent protozoa associated with waterborne parasitic outbreaks. The intestine is the target of three of the species most frequently found in humans: C. hominis, C. parvum, and. C. meleagridis. Despite the recent advance in genome sequencing projects for this apicomplexan, a broad genomic comparison including the three species most prevalent in humans have not been published so far. In this work, we downloaded raw NGS data, assembled it under normalized conditions, and compared 23 publicly available genomes of C. hominis, C. parvum, and C. meleagridis. Although few genomes showed highly fragmented assemblies, most of them had less than 500 scaffolds and mean coverage that ranged between 35X and 511X. Synonymous single nucleotide variants were the most common in C. hominis and C. meleagridis, while in C. parvum, they accounted for around 50% of the SNV observed. Furthermore, deleterious nucleotide substitutions common to all three species were more common in genes associated with DNA repair, recombination, and chromosome-associated proteins. Indel events were observed in the 23 studied isolates that spanned up to 500 bases. The highest number of deletions was observed in C. meleagridis, followed by C. hominis, with more than 60 species-specific deletions found in some isolates of these two species. Although several genes with indel events have been partially annotated, most of them remain to encode uncharacterized proteins.

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