The introduction of genomic approach to molecular biology has radically transformed today's experimental biology, and opened the door to a more rapid and comprehensive exploration of the molecular workings of living systems. Genomics implies a genome-scale analysis, i.e. that the experimental observations comprise the whole DNA level (structural and metagenomics) as well as expression level (the expression of gene, micoRNA, protein: functional genomics; expression networks: systems biology) investigation of a particular living organism or population. Genomic applications primarily play a role in an enhanced understanding of gene functions, which: 1.) brings about an ongoing change of outlook in both molecular and cell biology; 2.) results in knowledge that can be utilized in applied research, principally in biomedicine, agriculture and forensic science.

Genomics is developing in an incredible pace in the world's most developed countries, of this the best example is the exploration of the human genome, which was initiated in the early 90s and completed in 2001. Then this much time was needed for the sequencing of 3 billion base pairs, in contrast, today, only 8 years later, due to a revolutionary new technology, this task can be solved during a few weeks. In the West, genomics has already made its way into everyday medical practice. In the USA, numerous companies have been established and offering genomic services that are also accessible for the general public.  Naturally, the conditions in Hungary and Eastern Central Europe (hereinafter Region) are completely different, but there is a high demand for genomic services, primarily from the part of research institutes, here as well.

Our institute intends to perform world standard research and offer services in this very area of genomics, which is currently still in its initial stage in the Region, but expected to meet a serious market in the future. To support this goal,   we have acquired a high-tech instrument, that is unique in the whole Eastern Central European region.

The New Generation Sequencers (NGS), launched in 2007, enable the technical implementation of today's leading genome-level analyses, the so-called ultra-high throughput sequencing. These instruments open the possibility for the analysis of various biological samples from the analysis of microbial  phylogenetic trees through the sequencing following chromatin immunoprecipitation (ChIP-Seq) and to the copy number analysis of genes (see below for details)

The Applied Biosystems SOLiD^TM 4.0 System (see picture)  operated by BAYGEN is  a revolutionary new genetic analyzer platform, which enables the mass parallel sequencing of clonally amplified bead-bound DNA fragments. The sequencing method is based on the successive ligation of dye-labeled oligonucleotides. The ultra-high performance (100 Gigabase sequence /run) and the unparalleled accuracy (99.94 %) coupled with wide aplicational flexibility ensure a unique system.

Technological Invention

 In the Region, there are methods available, which, though gave a huge momentum to the answering of various biological questions at their introduction, have some inadequacies that have been shed light upon since. The two most significant of such methods are based on microarray ("chip") and PCR card techniques.

The special characteristics of the microarray method increase the number of genes that can be analyzed simultaneously by several magnitudes, thus they are able to provide us with a huge amount of structural (nucleotide sequence) and functional (gene and microRNA expression) information. This technology enables the analysis of gene expression, protein-DNA interactions and genetic variations. e.g. inherited gene defects (single nucleotide polymorphism; SNP) or copy number variations. An important inadequacy of the method, however, is that it is based on nucleic acid hybridization, which is a significant limiting factor when designing experiments.

• a precondition of the design of an array is the knowledge of the sequence to be analyzed,

• the analysis of sequences with a high degree of similarity is virtually impossible due to non-specific binding - cross-hibridization.

• the reproducibility of the experiments significantly differs between laboratories, and most importantly between platforms: for reliable results, the researcher has to perform each experiment with at least three biological replicates.

The PCR card, by being based on TaqMan technology, produces much more reliable results in measuring gene expression, but the number of genes that can be analyzed simultaneously is 384 maximum.  A further insufficiency of both methods is that they can detect only gene segments, mRNAs, microRNAs, and SNPs that have already been identified; the identification of new SNPs and microRNAs is technically not feasible.

NGS technology has appeared as an alternative of the above mentioned methods and, exactly due to its ultra-high throughput capacity - i.e. a single experiment produces millions of sequences-, it is displacing the microrarray technique in an increasing pace. Amongst the various advantages of the NGS technology, the following three can be mentioned:a) knowledge of the sequences to be analyzed is not necessarily required; b) paralog sequences can easily be differentiated from each other and c) quantitation takes place  in a really quantitative basis. As a result of this latter parameter, the NGS sequencings, in contrast to microarray analyses, do not require the use of three biological replicates, therefore the cost of the experiment is significantly reduced. The application of NGS technology is advantageous primarily when a higher coverage is coupled with more reliable results, such as when measuring gene expression, genotyping, exploration of DNA-methylation pattern or identification of transcription factor binding site. Besides these applications, the NGS technology enables the mass identification of new SNPs, and microRNAs, but it is also suitable for the identification of hitherto unknown species. (for details see below). There is no other technology available that can provide with the latter three applications.

A Short Introduction to the Technology

The NGS technology is much more economical in terms of materials, and work-hours and, above all, significantly faster than traditional sequencing technologies.

During the preparation of samples, the single-stranded DNA to be read is cut into fragments whose length have been predetermined by using special instruments.

Then each of these fragments are "tagged" with a complementer piece of a few nucleotides, which tags fix the DNA strands to a bead and, at the same time, facilitate the binding of the enzyme responsible for the synthesis of the complementer strand. 

At this point the enzyme and the nucleotides are added to the system; the appropriate combinations of the latter are labeled with different fluorescent dyes.

The incorporation of the nucleotides is then detected with an exceedingly sensitive camera, which registers as images the fluorescent signal emitted by the incorporating bases.  The observation of the images and the color flashes comprise the primary bioinformatical analysis, which can be monitored during the run, online. The camera, and the relevant data processing system is able to register simultaneously the sequences of several million DNA fragments in a single "screen field" in contrast to traditional sequencing methods, where only one DNA fragment is read at a given time: this is the very technology which ensures the ultra-high throughput capacity. The reverse translation of the color code to bases - secondary bioinformatical analysis - corresponds to the complementer sequence of the original DNA strand.  Finally, the data set of gigabase magnitude has to be interpreted: this is the tertiary bioinformatical analysis, which most of the time is performed by using specific softwares suitable for the given applications.

Consequently, during the various application of NGS technology the quality of the bases can be read individually, thus a significant enhancement in reading accuracy is achieved. A further advantage of this technology is the small final volume of the reactions, thus the quantities of the reagents are significantly reduced. Consequently, more accurate data can be gained with smaller material investment and in a significantly shorter time-frame. While the realization of the Human Genome Project took about ten years and required the investment of approximately 300 million USD, by using the NGS technology, a human genome can be determined in approximately 2 months and the costs are less than 10 thousand USD.

Areas of Application

Due to their high-throughput capacity, the new generation sequencers are suitable for a wide range of analyses:

• resequencing (sequencing of species with known reference genome e.g. phylogenetic classification of bacteria, the extremely accurate identification of sequence variations and SNPs)

• transcriptome analysis, gene-expression (RNA-Seq; Digital Gene Expression Profiling, DGEP, Whole transcriptome Analysis, WTA, 3' SAGE, 5' SAGE)

• de novo sequencing (sequencing of species with unknown genome)

• metagenomics, population genomics

•  the determination of haplotype

•  micorRNA-expression, identification

• sequencing following chromatin-immunoprecipiatation (ChIP-Seq; identification of transcription factor binding sites, mapping of active and passive chromatin.)

• DNA methylation analysis (Meth-Seq)

One or more of the techniques listed above are used by virtually every research, diagnostic or forensic laboratory, thus the number of potential users or customers is almost inestimable. This is exactly why NGS technology can play a dominant role in the further development of medicine, scientific research, criminal investigation, and all scientific fields that use practical applications for the investigation of the genetic material. Needless to say, the accurate knowledge of the whole DNA-sequence is invaluable, and it can be used for:

• the detection of genetic predisposition to diseases

• the exploration of therapeutic possibilities (personalized medicine)

• the diagnostics, prognostication of infections

• the identification of pathogens

• the determination of the degree of relatedness between two individuals or two organisms (e.g. determination of paternity, phylogenetics)

• personal identification (e.g. in criminal investigations)

• the development of vaccines

• breeding

• the comparison of gene variations

Practical Examples

Personalized medicine

Human genomes are  99,8 % identical; the remaining 0,2 % difference of the DNA  sequences between different individuals accounts for the extraordinary variability. This is why the 100% coverage is of utmost importance, because only in this case can we hope that, for example, predisposition to diseases, or factors responsible for drug resistance can be identified individually. Considering the fact that the total human genetic material consists of 3 billion base pairs, whose sequencing, by using traditional methods, would take years and several hundred million forints, it is rightly hoped that NGS technology can bring about a breakthrough in this area as well. It has a major significance mainly from the clinical point of view, since the individual genome patterns, predispositions to diseases as well as the understanding of the correlation between the efficacy of pharmaceuticals can greatly contribute to the advancement of personalized medicine. The very goal of pharmacogenomics, that is expected to undergo an abrupt development, is the development and application of personalized pharmaceutical treatments/therapies.

Reading of Repeated Sequences

The DNA of eukaryotic organisms contains repated sequences. These repeated sequences (repeats) can be located on the DNA molecule side by side, but they can be separated by a large (genetic) distance. The size and the number of repeated sequences show a very great variation among species, and in some cases they can constitute a significant portion of the genome. Some of the shared feature of these sequences are that they do not encode proteins, their origin has not been elucidated, and even their identification has not been completed.

The reading of the base sequence of long repeats has always been cumbersome because none of the above described, currently used methods enable the accurate reading and fitting of a long repeat. One of the features of the NGS technology, however, is the individual sequence-separation technique: following the reading of bases, the sequence-fragments that are located up to 10 kilobase apart can also be placed into the linear genome.  Practically, this means that a relatively small -3-4 megabase - procaryotic genome can be assembled into a few, or even a single coherent DNA sequence, a so-called "contig".

The Prognostication of  Predisposition to Tumorous Transformation

Regardless of the fact that the mechanism of development of a significant number of tumors is hitherto unknown - and the therapeutic procedures are also undeveloped -, a great number of gene defects are known to be in causal relation to the development of tumors. By using the NGS systems, and analyzing the appropriate DNA sequences, these gene defects can be mapped rapidly; the sequencing of the whole genome is unnecessary. Thus, the development of numerous tumors can be prevented/impeded and, at the same time, the therapeutic possibilities for the already existing tumors can be devised in a simpler, personalized way.

Trends and Prospects

Hungary has an internationally recognized scientific culture, and traditionally strong intellectual and researcher resources.  We are -still-  incomparably more competitive in the international scientific market than we are in the agrarian, industrial, or other markets. Consequently, science is our country's most efficient world market sector. However, as a result of the decrease in R&D&I investments, research places suffered significant losses during the transformation process, the condition of the instrumentation has deteriorated, the level of instrument supply and technical infrastructure is currently low. The technical infrastructure, instrumentation and tools of higher education and research institutes do not facilitate an equal opportunity participation in EU cooperation structures, and do not enable them to meet tender conditions.

Currently, the prevailing trends in the world's research and development show that development is increasingly dependent on the knowledge realized in advanced technical products and innovative services. As a result, the medium-term future of a country's research is significantly influenced by how much it invests in instrumentation, technical development and what course it chooses for its technical and technological policy.

Based on the world market experiences of the past few years, it can be stated that the NGS systems, due to their pioneering technology, are spreading exceedingly fast.  Namely, because the applications that can be run on the NGS systems - by ensuring significantly cheaper and more reliable investigations - completely replace techniques, that are used in Hungary as well, such as micro-array based tests. As a result of the versatility of the applicable techniques, professionals working in the areas of biological and medical sciences can all be mentioned as potential customers. Since, in comparison to the methods applied in today's medical science, the sequence determination of NGS systems ensures more expensive investigations, currently mainly researchers use them. The costs of the investigations, however, are expected to decrease as the technique is becoming more and more widespread, and medical and forensic science will certainly become an everyday user of the technique in a few years, thus an expansion of the market is expected. It is absolutely worth mentioning that more well-off private persons in the USA already employ the genome-sequencing service provided by this instrument, thus creating the possibility for personalized therapies. One of the companies has already sequenced the genome of two world renowned scientists - James Watson and J. Craig Venter -, and, amongst other things, the sequencing of the Neanderthal man is near to completion. The more affluent microbiology laboratories ever more frequently identify mutant bacteria by sequencing their whole genome, as it is remarkably fast and requires incomparably less labor investment than traditional methods do.

Service

The appearance of NGS systems has fundamentally altered the approach to biological and clinical questions. The installation of SOLiD V4.0 System  in our Institute resulted in a pioneering technology, which had been available for the researchers of the Region only in western laboratories and service centers. Thus the platform ensures a unique possibility for the whole Hungarian research community. Furthermore, it is important to note that, since NGS instruments are not easily accessible even for Western-European research groups, the accessibility of such an NGS operating platform essentially facilitates the entering of Hungarian laboratories into international (mostly EU) tenders, and consortia.

Our Institute offers the services related to the NGS platform for its interested partners in the broadest possible range. These services, adapting to the demand of customers, include, besides sequence-generation, the fast and accurate interpretation of experimental data.

1) since tender conditions allow academic, university, and foundation research groups to employ external services for the realization of their research and the costs of these are allowable within the tenders, one form of cooperation is the inclusion of contract sequencing costs in the tender;

2) we are open to initiating conjunct projects, submitting conjunct tenders, and to collaboration;

3) the platform also undertakes contract sequencing without any further cooperation for partners having the necessary funds to cover the costs of the service;

Bioinformatics

An established strategy of a significant portion of the companies undertaking NGS contract sequencing is that, following the basic data analysis, they leave the detailed analysis of the experiments to the customer laboratory, which as often as not has to employ a bioinformatician and has to enhance its available computer resources. Naturally, these significantly increase the costs of the experiments. Since the platform operated by us has both the bioinformatical expertise and the necessary computer resources, we offer the possibility for complete data analysis and interpretation.

• Read more: Sequencing technologies — the next generation (pdf) >>>

• Brochure: Next Generation Sequencing (english)