Mediante esta metodología se determina el orden o secuencia de los nucleótidos o bases nitrogenadas: adeninas (A), citosinas (C), guaninas (G) y timinas (T) a lo largo de la molécula de ADN. Con las nuevas tecnologías de Secuenciación de Nueva Generación NGS, el costo y el tiempo requerido para la secuenciación han disminuido significativamente. Existen diferentes plataformas y protocolos basados en NGS según el objetivo del proyecto de secuenciación.
Mediante esta técnica se secuencia el ADN de manera global, incluyendo además de las regiones que codifican proteínas, aquellas regiones no codificadoras (intrones, espaciadores génicos y el ADN “basura”) y los genes que regulan la expresión genética.
Secuenciación automatizada del exoma: Se secuencia el exoma, la fracción del genoma constituida por los exones, estos representan las regiones codificadoras del ADN y contienen información para la biosíntesis de proteínas. Esta secuenciación permite centrar los recursos en aquellos genes con mayores probabilidades de afectar el fenotipo.
Se usa para la secuenciación de aquellos genomas para los cuales no existe una secuencia de referencia disponible en las bases de datos. Proporciona información útil para mapear genomas de nuevas especies o para completar la secuenciación de genomas de organismos conocidos. Se puede usar para la secuenciación del genoma completo y también para hacer Shotgun sequencing de todo el genoma.
Esta técnica constituye la forma más eficiente para secuenciar un fragmento largo de ADN. El método consiste en cortar al azar la molécula de ADN en pequeños fragmentos que son secuenciados de manera individual, y posteriormente las secuencias resultantes se ensamblan mediante programas bioinformáticos. Este proceso se repite una y otra vez, hasta lograr la obtención de la secuencia completa del ADN.
La secuenciación dirigida de próxima generación le permite secuenciar áreas específicas del genoma para análisis en profundidad de manera más rápida y rentable que la secuenciación del genoma completo (WGS). La secuenciación dirigida utiliza una secuenciación profunda para detectar variantes nuevas y conocidas dentro de su región de interés. Este método generalmente requiere menos cantidad de muestra y produce una menor cantidad de datos que WGS, lo que hace que los análisis sean más manejables. Es de utilidad para el estudio de la biodiversidad de nuestro planeta, para la detección de genes relacionados con enfermedades hereditarias y para monitorear el efecto del medio ambiente en nuestro genoma, entre otras.
Genes determine all the characteristics of living things. They determine, for example, that one variety is resistant to a pathogen or to salinity or cold and another is not. With RNA-seq, the sequencing of all the RNAs of the genes is carried out (at a given time and under different physiological conditions and with great sensitivity), this allows the discovery of new genes and rare genes with potential use for genetic improvement.
One of the most important applications of RNA-seq is to determine the differential expression of genes between two or more conditions. These can include treated versus not treated with some product, infected with a pathogen versus mock-inoculated, diseased versus healthy tissue, etc. The genes that are expressed for example in a disease resistant variety are not the same as those that are expressed in the susceptible one. RNA-seq quantifies how many times a gene is expressed in one condition relative to another. Once the genes associated with a certain trait of interest have been identified, they can then be used in the early selection of individuals with interesting agronomic traits in programs for the genetic improvement of plant or animal varieties.
RNA sequencing allows the identification of variants including SNPs, small insertions-deletions and structural variations, in addition to allele-specific expression. This is very useful in genetic improvement, and these variations can be used as:
This would save a lot of time in improvement programs, especially in long-cycle crops and for fruit characters or of late appearance. Similarly, SNPs and other variants can be used to construct molecular marker maps that are very useful when assembling the genome of a species for which there is no reference genome.
Without a doubt, commonly used DNA markers have contributed to mapping and association studies that led to the discovery of genes of interest. However, these DNA markers are randomly derived from polymorphic sites in the genome that may not be related to any traits of interest. Functional markers develop from polymorphic sites within genes that cause variation in phenotypic traits and are therefore more useful. RNA-seq is a great tool for the development of these markers.
The use of this service allows the identification and quantification of the expression of genes responsible for important characters in plants and animals, which is why it constitutes the basis for the identification of markers for molecular marker-assisted selection (MAS) that saves time and resources in plant and animal improvement programs.
When we perform RNA-seq of two contrasting genotypes for a trait of interest, it is possible to identify genes that are associated with this particular trait. On the other hand, RNA-seq is useful for the identification of candidate genes and SNPs useful for association mapping programs that are based on linkage disequilibrium or non-random association of alleles at different loci.
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RNA-seq is an NGS-based technique to determine the presence and quantity of messenger RNA in a given sample at a given time, therefore it is used to observe the continuous changes of the cellular transcriptome.
We guide you from the design of the project to the analysis of the results that you can access from our cloud or the information will be delivered to you. We also put our bioinformatics services at your disposal.
RNA-seq Applications:
A SNP, or single nucleotide polymorphism, is the substitution of one nucleotide for another at a certain position in the genome, as long as this substitution is found in at least 1% of the population.
SNPs are the most widely used markers today because they offer a greater number of advantages than other markers, such as their large number and wide distribution in the genome of all individuals that offers the possibility of building high-density maps required to isolate and studying genes of traits of interest and the possibility of using various automated genotyping methods.
bioSEQs offers different services for detection and validation of SNPs depending on the characteristics of the species in question and the objectives of your project, such as the development of SNPs from:
SNPs Applications:
In Spain, it is estimated that more than 50% of fruit plants; 30% of cereal seeds, and 20% of tomato seeds are reproduced without a breeder’s license. Molecular markers such as SNPs and others are not only a simple and cheap tool, but in several cases they constitute the only way to demonstrate varietal identity.
SNPs are currently the marker of choice to find association between molecular markers and traits of interest due to their high abundance and their distribution throughout the genome. One of the most used strategies is known as GWAS (genome wide association studies) in which it seeks to associate SNPs with important traits such as fat content in animals, milk production, weight gain or resistance or susceptibility to animal and plant diseases. These markers can be used in the early selection of these traits (molecular marker assisted selection).
Using SNP markers, it is possible to assess the quality of a pedigree or even infer it when genealogical data is not available.
Carrying out genetic evaluations is an important activity for the genetic improvement of livestock, because from them the genetic values are generated, which are an estimate of the productive merit of an animal for one or more productive characteristics and that help producers to make selection decisions. The information of genetic markers allows to increase the accuracy in the estimation of genetic values.
A QTL, or quantitative trait loci, is a region of DNA that generally contains several genes responsible for a trait. Many are the efforts that are made to find markers associated with these regions for the most important characters in plants and animals and to use them for their selection. SNPs, due to their great abundance and distribution, are an appropriate marker for QTL mapping.
Whole Genome Sequence (WGS) provides the most complete information about a microorganism. The genome can be analyzed using different WGS identification techniques, such as single nucleotide polymorphisms (SNPs) or Whole Genome Multi Locus Sequence Typing (wgMLST) sequencing typing to identify the viruloma, resistome and the antibioresistome.
The WGS of microorganism genomes holds great promise for improving pathogen diagnostics, as well as screening of industrial, agricultural, and environmental strains. Furthermore, this technique is very useful for comparative genomics, that is, differentiations between strains or typing of strains.
The registration of biofertilizers in many countries requires the submission of proof that the microorganisms they contain have been identified by a sequencing technique. Therefore, scientifically proven and state-of-the-art techniques should be used.
Ecological genomics seeks to understand the genetic mechanisms related to the response of organisms to their natural environments. Its objective is to understand how the genes of organisms have been influenced by their natural environments. Current technological advances, especially advances in sequencing techniques, help to connect genetic information with ecological studies.
The rapid and correct identification of pathogens is an essential requirement for the diagnosis and subsequent application of a suitable treatment, whether in humans or animals or even plants. At present, many of the pathogenic microorganisms can be easily identified by conventional microbiological techniques, which require prior isolation of the pathogen and are based on phenotypic characteristics. However, when it comes to new pathogens never before described or phenotypically indistinguishable from other similar ones, identification and typing by genome sequencing is necessary. A recent example is the coronavirus.
When it comes to identification, reference is made to the classification of the organism in question within an already established group or taxon, for its part, the typification refers to a more specific classification, identifying variants that even allow knowing the subspecies. Genome sequencing allows deeper typing, being able to offer information such as antibiotic resistance genes, virulence genes, etc.
This is necessary in the following cases:
1- Identification of strains with little description, low frequency of isolation, or phenotypically atypical.
2- Identification of microorganisms that are not cultivable or difficult to culture.
3- Identification of microorganisms whose biochemical characteristics are not adapted to those of any recognized genus or species, which can sometimes go as far as the description of new pathogens.
4- Taxonomic or phylogenetic studies for epidemiological purposes.
Identification and typing applications by whole genome sequencing:
DNA barcoding is a species identification method that uses a short segment of DNA from a specific gene or genes (ITS, 16S or 18S). It is based on the premise that one of these individual sequences can be used as the barcode of the species (ITS Sequence of a fungus) and compared to a library of those sequences, to identify the species in the same way as scanners of supermarkets identify products by their barcode. Metabarcoding is a large-scale taxonomic identification of complex environmental samples that is based on high-throughput sequencing technologies such as Illumina and Oxford Nanopore Technologies, available from our company.
This technology enables researchers to explore microbiome and environmental samples at a much deeper level than was previously possible.
The identification of bioindicator organisms such as invertebrates, bacteria, plants, etc., is important for studying the health of ecosystems.
For example, the use of aquatic macroinvertebrates (and especially insects) as indicators of the quality of water in ecosystems has become widespread in recent years.
Traditionally the identification and monitoring of these species was done with morphological indicators, which presents many limitations.
Metabarcoding has the potential to overcome the limitations of species identification methods based on morphology.
In a similar way, this technique can be applied for the identification, classification and study of populations of pests that affect crops, and consequently humans and animals. For example, it is very useful for the identification of species which cannot be diagnosed on the basis of their morphology. In holometabolic insects, the finding of eggs, larvae and pupae dissociated from the adult specimens hardly allows adequate identification, so “barcoding” is a very useful tool to intercept invasive species that can become pests in different countries.
Barcoding is useful both for the verification of raw materials that are used in the preparation of food individually and for the verification of all the species present in foods made with complex mixtures, such as highly processed foods that have lost their morphological features, and thus prevent food fraud.
This method can be routinely implemented to check raw materials, traceability, and to detect adulterations or contamination along supply chains or to certify the finished product.
Barcoding can be useful in detecting fraud such as:
It can also be used to:
Microorganisms are introduced into food from raw materials and human beings are capable of growing and surviving in a processing environment.
Cleaning and environmental control practices are normally established to control contaminating microorganisms.
On many occasions, those microorganisms responsible for the deterioration of food and the origin of the pathogenic microorganisms are not known.
Through the use of DNA metabarcoding, the identity of all microorganisms present in food processing chains can be determined, which makes this technique a powerful tool in spoilage investigations, shelf life analysis and environmental control.
This technique will offer you several advantages over traditional methods of studying food contaminating microorganisms:
On those occasions in which laboratories keep a stock of bacterial species, fungi or microorganisms in general, either to carry out certain studies with them or for their use as bioproducts, it is necessary to maintain their purity. Morphological classifications are often not sufficient due to the similarity that often exists between different species (for example cryptic or twin species).
We offer a stock homogeneity testing service to confirm that your preserved samples actually belong to the species in question and are pure.
We analyze a significant number of individuals from your stock using DNA barcoding techniques to give a reliable diagnosis of their genetic purity.
To manage and conserve biodiversity, it is first necessary to make an accurate estimate of the species present in a given ecosystem.
That way you can follow up and know if any species is being lost, where and why, and what measures to avoid it could be more effective.
Metabarcoding technology can characterize the species composition of complex environmental samples in a reliable and cost-effective way, making this technique a powerful tool for conducting biodiversity studies.
Metabarcoding offers several advantages over traditional methods:
The term “microbiota” refers to the set of commensal, symbiotic and pathogenic microorganisms that live in a common environment, while the term “microbiome” refers to the collection of all their genomes. This includes bacteria, fungi, viruses, protists, and archaea.
The portion of the soil influenced by the roots of the plants, the rhizosphere, constitutes a niche of great microbial diversity, strongly determined by the exudates of the plants.
The microorganisms of the rhizosphere, play important roles in plant nutrition and in adaptation to different types of stress.
Indeed, the microbiome of plants can represent an additional source of genes and functions for their host that can expand the possibilities of plants to adapt to environmental changes.
bioSEQs offers a soil sample metabarcoding service that allows you to identify, for example, all the microorganisms present in the rhizosphere of certain plants, either to identify possible microorganisms with potential use as biofertilizers, as well as pathogens and the interactions of beneficial organisms with pathogens.
The efficiency of various bioprocesses such as the production of biogas through anaerobic digestion or the treatment of wastewater, depend on the microbial composition at the different stages of the process.
For this reason, it is necessary to monitor the changes that occur in the composition of microorganisms since this can determine the success or failure of the entire process.
DNA metabarcoding is an excellent technique to monitor microbial communities and identify indicators of the good or bad performance of the process.
At bioSEQs we offer a specialized service for the monitoring of bacterial communities by metabarcoding and we provide you with a complete report that includes the details of the methods used, the sequence data and the species present in the sample and their relative abundance.
A number of microorganisms participate in the wine fermentation process, some of which are desirable while others such as bacteria that produce acetic acid are considered contaminants. Rapid, specific, sensitive and reliable detection methods are needed to detect the microorganisms present in this process and to give it adequate monitoring. All these requirements are met by the qPCR.
Plating techniques have historically been used to isolate and identify bacteria and yeast, but this process is laborious and can lead to errors. qPCR solves this problem as many populations do not respond to plating, making it appear that they are not present.
qPCR protocols exist to detect Oenococcus oeni, lactic and acetic acid bacteria, Dekkera, Bruxellensis, S. cerevisiae, and Zygosaccharomyces species. The qPCR is superior to other methods since it not only allows to identify the microorganism but also to quantify the size of its population.
It should also be taken into account that the land where the vineyard is grown and its communities of microorganisms are very important when making wine, because they influence its final properties.
qPCR is an essential technique in the study of host-pathogen interaction.
For example, it can be used to detect the presence and quantify vascular fungi such as Verticillium in olive, tomato or potato, as well as diseases caused by different species of Fusarium or by Oomycetes.
It is especially useful for detecting diseases caused by viruses and viroids that are microscopic and cannot be cultured.
As an example, mention can be made of the citrus exocortis viroid, the apple mosaic virus, the infectious short internode virus of the vine, among others.
It is also an essential technique to detect bacterial diseases such as Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae in tomato, Xylella fastidiosa in olive, almond and apple trees and Pseudomonas syringae pv. phaseolicola in beans among others.
The technique is also used to evaluate the effect of control treatments on microorganism populations, to evaluate the activating effect of defense pathways (JA / ET and SA) of plants by resistance inducers and to evaluate the differential expression of genes in the presence of pathogens in resistant and susceptible cultivars and identify genes with potential use in breeding.
Scientists also use it to determine whether a gene has been correctly silenced by a gene silencing technique such as VIGS or to monitor the effect of certain treatments on gene expression.
Similarly, it can be used to evaluate the activation of routes of induced systemic resistance (ISR) by soil microorganisms or acquired systemic resistance (SAR) by commercial inducers such as Acibenzolar.
qPCR, or quantitative PCR, is the most widely used and reliable technique to detect and determine the abundance of microorganisms such as viruses, bacteria, archaea, fungi, and protists in plant or animal tissue samples, soil samples, and other environmental samples.
It is highly specific as it is based on the detection of a nucleic acid segment of the species under study, and it is also a rapid and cost-effective assay.
To quantify the amount of microorganisms present in a given sample, calibration curves are generated from known concentrations of plasmid or genomic DNA. By running our samples and comparing them with this curve, the number of nucleic acid molecules present in the samples of interest can be quantified.
The process consists of the following main stages:
Microsatellites, or Simple Sequence Repeats (SSRs), are motifs of 1-6 adjacent repeating nucleotides that are found distributed in the genome in both coding and non-coding regions.
The microsatellite technique consists of the PCR amplification of these hypervariable regions using primers for specific regions surrounding the microsatellites.
Its advantages include being relatively abundant, the high level of allelic variation, presenting codominant inheritance, analytical simplicity, and the repeatability of results between laboratories.
With the advent of sequencing techniques, the range of species for which there are primers available to perform this molecular marker technique has expanded.
Its simplicity and high reproducibility make it especially useful for the characterization of clones and varietal identification.
bioSEQs offers a varietal identification service with microsatellite markers for various plant species.
The process consists of the following stages: