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

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.

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

Genetics ◽

10.1534/genetics.120.303553 ◽

2020 ◽

Vol 216 (2) ◽

pp. 343-352

Author(s):

Denis Touroutine ◽

Jessica E. Tanis

Keyword(s):

Caenorhabditis Elegans ◽

Low Cost ◽

Point Mutations ◽

Primer Design ◽

Design Parameters ◽

Amplification Efficiency ◽

Single Nucleotide ◽

Sequence Composition

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.

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 ◽

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.

Distribution of Fitness and Virulence Effects Caused by Single-Nucleotide Substitutions in Tobacco Etch Virus

Journal of Virology ◽

10.1128/jvi.00524-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 12979-12984 ◽

Cited By ~ 136

Author(s):

Purificación Carrasco ◽

Francisca de la Iglesia ◽

Santiago F. Elena

Keyword(s):

Single Case ◽

Site Directed Mutagenesis ◽

Relative Fitness ◽

In Planta ◽

Phenotypic Effect ◽

Single Nucleotide ◽

Mutant Genotype

ABSTRACT Little is known about the fitness and virulence consequences of single-nucleotide substitutions in RNA viral genomes, and most information comes from the analysis of nonrandom sets of mutations with strong phenotypic effect or which have been assessed in vitro, with their relevance in vivo being unclear. Here we used site-directed mutagenesis to create a collection of 66 clones of Tobacco etch potyvirus, each carrying a different, randomly chosen, single-nucleotide substitution. Competition experiments between each mutant and the ancestral nonmutated clone were performed in planta to quantitatively assess the relative fitness of each mutant genotype. Among all mutations, 40.9% were lethal, and among the viable ones, 36.4% were significantly deleterious and 22.7% neutral. Not a single case of beneficial effects was observed within the level of resolution of our measures. On average, the fitness of a genotype carrying a deleterious but viable mutation was 49% smaller than that for its unmutated progenitor. Deleterious mutational effects conformed to a beta probability distribution. The virulence of a subset of viable mutants was assessed as the reduction in the number of viable seeds produced by infected plants. Mutational effects on virulence ranged between 17% reductions and 24.4% increases. Interestingly, the only mutations showing a significant effect on virulence were hypervirulent. Competitive fitness and virulence were uncorrelated traits.

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 ◽

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.

Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis studies

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2010.0063 ◽

2010 ◽

Vol 365 (1548) ◽

pp. 1975-1982 ◽

Cited By ~ 110

Author(s):

Rafael Sanjuán

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Single Nucleotide ◽

Single Stranded Dna ◽

Dna Viruses ◽

Fitness Effects ◽

Lethal Mutations ◽

Mean Fitness ◽

The Mean

The fitness effects of mutations are central to evolution, yet have begun to be characterized in detail only recently. Site-directed mutagenesis is a powerful tool for achieving this goal, which is particularly suited for viruses because of their small genomes. Here, I discuss the evolutionary relevance of mutational fitness effects and critically review previous site-directed mutagenesis studies. The effects of single-nucleotide substitutions are standardized and compared for five RNA or single-stranded DNA viruses infecting bacteria, plants or animals. All viruses examined show very low tolerance to mutation when compared with cellular organisms. Moreover, for non-lethal mutations, the mean fitness reduction caused by single mutations is remarkably constant (0.10–0.13), whereas the fraction of lethals varies only modestly (0.20–0.41). Other summary statistics are provided. These generalizations about the distribution of mutational fitness effects can help us to better understand the evolution of RNA and single-stranded DNA viruses.

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 ◽

High Resolution Melt ◽

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.

Distinct genetic spectrums and evolution patterns of SARS-CoV-2

10.1101/2020.06.16.20132902 ◽

2020 ◽

Cited By ~ 5

Author(s):

Sheng Liu ◽

Jikui Shen ◽

Lei Yang ◽

Chang-Deng Hu ◽

Jun Wan

Keyword(s):

Complete Genome ◽

Clustering Method ◽

Genome Sequences ◽

High Quality ◽

High Coverage ◽

Single Nucleotide ◽

Multiple Groups ◽

Over Time

AbstractFour signature groups of single-nucleotide variants (SNVs) were identified using two-way clustering method in about twenty thousand high quality and high coverage SARS-CoV-2 complete genome sequences. Some frequently occurred SNVs predominate but are mutually exclusively presented in patients from different countries and areas. These major SNV signatures exhibited distinguished evolution patterns over time. Although it was rare, our data indicated possible cross-infections with multiple groups of SNVs existed simultaneously in some patients, suggesting infections from different SARS-CoV-2 clades or potential re-combination of SARS-CoV-2 sequences. Interestingly nucleotide substitutions among SARS-CoV-2 genomes tend to occur at the sites where one bat RaTG13 coronavirus sequences differ from Wuhan-Hu-1 genome, indicating the tolerance of mutations on those sites or suggesting that major viral strains might exist between Wuhan-Hu-1 and RaTG13 coronavirus.

High-throughput genotyping assay for the large-scale genetic characterization ofCryptosporidiumparasites from human and bovine samples

Parasitology ◽

10.1017/s0031182013001807 ◽

2013 ◽

Vol 141 (4) ◽

pp. 491-500 ◽

Cited By ~ 12

Author(s):

J. L. ABAL-FABEIRO ◽

X. MASIDE ◽

J. LLOVO ◽

X. BELLO ◽

M. TORRES ◽

...

Keyword(s):

High Throughput ◽

Sanger Sequencing ◽

Large Scale ◽

Genetic Characterization ◽

Low Cost ◽

Cost Effective ◽

Single Nucleotide ◽

Identification Of Species

SUMMARYThe epidemiological study of human cryptosporidiosis requires the characterization of species and subtypes involved in human disease in large sample collections. Molecular genotyping is costly and time-consuming, making the implementation of low-cost, highly efficient technologies increasingly necessary. Here, we designed a protocol based on MALDI-TOF mass spectrometry for the high-throughput genotyping of a panel of 55 single nucleotide variants (SNVs) selected as markers for the identification of commongp60subtypes of fourCryptosporidiumspecies that infect humans. The method was applied to a panel of 608 human and 63 bovine isolates and the results were compared with control samples typed by Sanger sequencing. The method allowed the identification of species in 610 specimens (90·9%) andgp60subtype in 605 (90·2%). It displayed excellent performance, with sensitivity and specificity values of 87·3 and 98·0%, respectively. Up to nine genotypes from four differentCryptosporidiumspecies (C. hominis, C. parvum, C. meleagridisandC. felis) were detected in humans; the most common ones wereC. hominissubtype Ib, andC. parvumIIa (61·3 and 28·3%, respectively). 96·5% of the bovine samples were typed as IIa. The method performs as well as the widely used Sanger sequencing and is more cost-effective and less time consuming.

FastGT: an alignment-free method for calling common SNVs directly from raw sequencing reads

10.1101/060822 ◽

2016 ◽

Author(s):

Fanny-Dhelia Pajuste ◽

Lauris Kaplinski ◽

Märt Möls ◽

Tarmo Puurand ◽

Maarja Lepamets ◽

...

Keyword(s):

Y Chromosome ◽

Low Cost ◽

Source Code ◽

Computational Method ◽

Single Nucleotide ◽

Link Type ◽

Genome Data ◽

Alignment Free

We have developed a computational method that counts the frequencies of unique k-mers in FASTQ-formatted genome data and uses this information to infer the genotypes of known variants. FastGT can detect the variants in a 30x genome in less than 1 hour using ordinary low-cost server hardware. The overall concordance with the genotypes of two Illumina “Platinum” genomes1 is 99.96%, and the concordance with the genotypes of the Illumina HumanOmniExpress is 99.82%. Our method provides k-mer database that can be used for the simultaneous genotyping of approximately 30 million single nucleotide variants (SNVs), including >23,000 SNVs from Y chromosome. The source code of FastGT software is available at GitHub (https://github.com/bioinfo-ut/GenomeTester4/).

Low-cost and clinically applicable copy number profiling using repeat DNA

10.1101/394429 ◽

2018 ◽

Cited By ~ 1

Author(s):

S Abujudeh ◽

SS Zeki ◽

MCV van Lanschot ◽

M Pusung ◽

JMJ Weaver ◽

...

Keyword(s):

Copy Number ◽

Large Scale ◽

Low Cost ◽

Clinical Use ◽

Copy Number Alterations ◽

Single Nucleotide ◽

Repeat Dna ◽

Gene Panels ◽

Somatic Copy Number Alterations

AbstractLarge-scale cancer genome studies suggest that tumors are driven by somatic copy number alterations (SCNAs) or single-nucleotide variants (SNVs). Due to the low-cost, the clinical use of genomics assays is biased towards targeted gene panels, which identify SNVs. There is a need for a comparably low-cost and simple assay for high-resolution SCNA profiling. Here we present our method, conliga, which infers SCNA profiles from a low-cost and simple assay.