Services

This page lists the different DNA sequencing services:

Whole Genome Sequencing

Genome sequencing is the determination of the entire DNA sequence in the cell nucleus and mitochondria. In contrast to exome sequencing (determination of the sequence of only protein-coding regions – exons and splicing sites), the method allows one to “read” other DNA regions that do not directly carry information about the protein but regulate its production.

Despite the fact that there are more than 6,000 genetic nosologies caused by known changes in the DNA sequence, in many patients the diagnosis remains unconfirmed by the molecular genetic method.

Many genetic diseases are clinically similar to each other, but they are caused by mutations in completely different genes. Also, the causes of diseases can be mutations in non-coding regions and in mitochondrial DNA.

Today, most genetic tests are aimed at finding mutations in one or more genes. Small steps to expand the studied genes can significantly delay the moment of diagnosis.

Genome sequencing can significantly accelerate the diagnostic search due to the ability to accurately detect not only single-nucleotide pathogenic variants, but also insertions / deletions, copy number variations, and other changes in the DNA sequence.

Сapabilities:

• Determination of variants with high accuracy, including in non-coding regions of the genome;
• Gene copy number variations (CNV), such as deletions and duplications of various sizes;
• Balanced chromosomal changes with the definition of break points;
• Areas of loss of heterozygosity and homogeneous disomy;
• Variants in the mitochondrial genome with heteroplasmy detection> 5%;
• Expansion of tandem repeats in 37 genes.

Indications:

• First-line study if differential diagnosis is required among a heterogeneous group of hereditary diseases (clinically similar), with nonspecific combinations of congenital malformations, in the presence of which it is difficult to choose a targeted study;
• Study of the second line, with a negative result of other diagnostic methods;
• Search for mutations in non-coding regions of the genome.

Full genome sequencing as a first-line study, thanks to a professional algorithm that meets ACMG requirements, allows you to get accurate results, shorten the time to diagnose a hereditary disease, and, in most cases, is the most cost-effective method.

De Novo Whole Genome Sequencing

De Novo Whole Genome Sequencing is a method of assembling transcriptome sequences, which is carried out without mapping to a reference genome. Individual RNA sequences or transcripts are reconstructed from short fragments (reads or reads) obtained during sequencing.

De novo assembly of a transcriptome does not require a reference genome. Considering that most organisms have not yet been sequenced, the de novo assembly of the transcriptome of such organisms can be used as the first stage in their study. For example, for the study and comparison of transcriptomes between oranisms, as well as for the analysis of differential expression under various effects on the body. Sometimes it is useful to assemble a de novo transcriptome, even if a reference genome is present, as this can detect transcribed regions whose sequences are not present in the genomic assembly. Having a de novo assembly and a reference genome, transcripts of exogenous origin can be detected. The most important difference between de novo assembly is that it does not require sequence alignment and solving problems of finding or predicting splicing sites; in addition, it is possible to collect transcripts obtained as a result of trans splicing.

However, de novo assembly is an algorithmically complex and computationally expensive process. Also, this approach is highly sensitive to errors.

Whole-Exome Sequencing

Whole-exome sequencing includes the analysis of the coding sequences of all known genes.

Clinical exome sequencing is the optimal study for suspected hereditary diseases in terms of the ratio of the likelihood of finding clinically significant mutations and research costs. 85% of all known clinically significant mutations are contained in it.

Sequencing – establishing the nucleotide sequence of DNA, the most accurate analysis of hereditary information to date. The genetic structure of DNA determines the presence of genetic (hereditary) diseases. There are more than 4800 genes available.

Benefits of the technique:

• Sequencing is done once in a lifetime. Re-examination is not required;
• During sequencing, the patient and the doctor receive all the necessary information about the structure of genes, which eliminates the need for additional genetic testing and uncertainty in the diagnosis;
• The advantage of the study “Whole exome sequencing” over the analysis of the entire genome is that it allows a massive and one-time screening of the most important, i.e. protein-coding sequences.

Indications:

• if you suspect a genetic pathology for which there are no specialized genetic tests;
• in the case of a search for mutations in genes for which there are no specialized genetic tests;
• if necessary, comprehensive genetic testing in case of suspicion of genetic pathology;
• with an unclear clinical picture of the disease in the case when the exact choice of genes necessary for analysis is difficult;
• when determining the carriage of monogenic diseases when planning a child;
• for the assumption or confirmation (but not exclusion!) of the clinical diagnosis in controversial cases;
• to clarify the prognosis of the course of the disease;
• to clarify the hereditary genetic risk for family members.

Preparation:

Genetic testing does not require special training. For the study, blood is taken in the morning on an empty stomach. It is recommended to take blood no earlier than 4 hours after the last meal.

Before the diagnosis, it is not recommended to expose yourself to stressful situations, take alcoholic beverages and smoke. Diet and medications do not affect the performance of the test.

Custom Targeted Sequencing

Custom targeted sequencing allows you to focus on specific regions of the genome or individual genes. This method is used when a patient has a suspicion of a genetic disease with a known molecular etiology. In such cases, it is sufficient to establish the structure of only one gene or some of its regions, the so-called hot spots, where the presence of a mutation is most likely.

For custom targeted sequencing, the SANGER method is usually used, which allows you to establish the sequence of small DNA fragments – up to 1000 nucleotides in one study. This method is quite suitable for the analysis of one or more small genes.

Sequencing cDNA or individual exons is the most efficient way to detect mutations. Quite often, the primary search for violations in the coding regions of a gene is carried out in this way. For genes that are relatively small (for example, the coagulation factor IX gene for hemophilia B), direct sequencing is sometimes used as the primary method for scanning for mutations.

However, despite the availability of techniques (various modifications of PCR, the use of mRNAll to obtain cDNA), making sequencing a routine method, sequencing of full-length cDNA (i.e., all exons) to identify mutations in individual individuals remains laborious, expensive and costly. time by the procedure. Therefore, in practice, DNA fragments obtained by amplification or cloning are pre-tested for mutations by simpler methods based on a comparison of the physicochemical characteristics of mutant and normal sequences. However, the exact molecular characteristics of each mutation, regardless of its nature (nucleotide substitutions, deletions, duplications, insertions, etc.), can be obtained only by direct sequencing.

Benefits:

• Allows focusing on specific areas, making it much more convenient to analyze the result;
• Significantly reduces costs (time and money);
• Through deep sequencing at different levels, information on rare deviations can be obtained;
• Affordable technique.

Method availability:

Compared to complex analysis methods, targeted sequencing is more beneficial. Only certain parts of genes are investigated in which mutations can be determined. In this case, fewer resources are required for diagnostics, so a targeted search for individual deviations will be much cheaper than full diagnostics.

Bisulfite Sequencing

Bisulfite sequencing is a technique in which different regions of DNA are analyzed using methylation. Methylation is the process of adding a specific molecule called a methyl group to a nucleotide, in this case usually cytosine. Inactive nucleotides are often methylated, so this method can be used for a variety of purposes, from identifying active regions of the genome to identifying gene-rich regions. In bisulfite sequencing, methylated cytosines do not interfere with the sequencing process, while unmethylated cytosines are converted to uracil, a nucleotide not normally found in genetic material, deoxyribonucleic acid (DNA).

This method is very sensitive to changes in methylation, so small changes in binding can give researchers specific information about specific nucleotides. Sodium bisulfite converts cytosine to uracil, but conversion occurs in an environment where methylated cytosine will not undergo this change. When the bisulfite sequencing was complete, the original DNA was transformed into a substantially different molecule. Cytosines will be severely depleted or potentially absent. If cytosine is still in this converted molecule, it is methylated cytosine naturally in the genome in question.

As with all experimental protocols, bisulfite sequencing has disadvantages. Its most significant disadvantage is that it requires a very high salt concentration to function properly. Salt aids in the annealing of single-stranded DNA into its more natural double-stranded helix, and sodium bisulfite cannot always reach cytosines when they are part of the double-stranded DNA. If the salt concentration is too high, a number of cytosines may not be converted to uracil, which leads to false identification of methylated cytosines in the genome. Denaturing agents may be needed to minimize false positives.

Large amounts of genomic data are not needed for bisulfite sequencing, so this method has useful applications for the analysis of clinical samples. The original source of the nucleic acid is irrelevant, but the source must be DNA. In theory, ribonucleic acid (RNA) can be sequenced using this method, since most of the RNA is single stranded and would not be as susceptible to false positives due to blocked nucleotides. However, in practical applications, bisulfite sequencing is useless for RNA, since it naturally contains uracil. Without any external labeling or addition to the protocol, the converted cytosines would be indistinguishable from natural uracil.

When undertaking any type of sequencing methodology, accuracy and precision are essential. Sensitive techniques, such as bisulfite sequencing, offer a robust sequencing tool that, in turn, allows gene analysis and targeting of drugs and therapies. While this method cannot be used on living people, it can still only be very useful for the smallest tissue samples to work with.

ChIP-seq

ChIP-seq is a DNA-protein interaction analysis method based on chromatin immunoprecipitation (ChIP) and high-performance DNA sequencing. The method was developed to study histone modifications throughout the genome, as well as to search for binding sites for transcription factors. Previously, the most popular method for establishing DNA-protein interactions was ChIP-on-chip, which combines chromatin immunoprecipitation with hybridization on DNA microarrays.