Genomic Laboratory offers next generation sequencing on two Illumina platforms – MiSeq and HiSeq. Furthermore, it provides library generation services in the Access Array (Fluidigm) system and performance of emulsion PCR reaction.
The comprehensive management of advanced equipment of the Workshop was entrusted to the specialists of Centrum Badań DNA sp. z o.o.
Next generation sequencing (NGS) consists of simultaneous synthetic sequencing of cloned or single DNA particles on a single microchip (flow cell). NGS sequencing uses prolonging iteration cycles with polymerasis of the DNA matrix chain and detachment of the marked terminator. In each cycle four nucleotides are attached, each of them marked with a different terminator colour (each cycle – prolonging of the chain by 1 alkali)
Next generation sequencing allows for precise reading of sequence at an accuracy of above 99.9% per single alkali which surpasses the potential of any other laboratory method, and provides insight into the genetic information in form of genomes, transcriptomes or epigenomes of any organisms.
NGS Illumina platforms: MiSeq and HiSeq.
The Workshop offers comprehensive genome NGS sequencing services, starting from material isolation (out of tissue, leukocyte sediment, blood, swab), by purification, quantitative fluorescence (Qubit), preparing of libraries, post-sequencing and biodata analysis.
- Genome analysis (sequencing of entire genome, de novo sequencing, de novo sequencing of small genomes (bacteria, yeast, viruses) or plasmides, Mate Pair sequencing, exome sequencing, targeted resequencing, amplicon sequencing),
- Transcriptome analysis (RNA – RNAseq sequencing, miRNA sequencing)
- Epigenome analysis (Analysis of interaction between DNA and protein (Chip-Seq), methylation analysis)
- Expression microarrays
- Metagenomics (16S)
- Genetic panels (cancer panels, cardiomyopathies, autism, entire spectrum of innate genetic diseases)
De novo sequencing
A unique combination of reading length and depth, combined with the possibility of paired sequencing of inserts of varying length makes the MiSeq platform a perfect de novo sequencing solution of small genomes. The reading accuracy of raw data generated by the system enables the skilful and effective formation of high-quality contigs and the identification of biologically relevant variants.
Sequencing of amplicons
The sequencing of amplicons is surely an advantageous feature of the MiSeq platform. In opposite to the traditional Sanger sequencing method providing a double coverage at the highest, NGS sequencing provides a coverage of few hundred thousands depending on the project complexity and needs. As a rule, a coverage of 20-30x is adopted which means that the given DNA alkali is read 20-30 times.
Metagenomics (16S RNA)
Metagenomics aims at the definition of the quantity and type of microorganisms in a sample. Metagenome surveys are commonly used for the analysis of the procariotic gene 16S rRNA which is approx. 1 500 bp long, consisting of nine alternate conservative and fluctuating regions, which facilitate sequencing and filogenetic classification of any microorganism species out of a complex population of microorganisms.
The MiSeq system is the most complex and cost-efficient solution for all metagenome research. It allows for the mapping of an entire genome of specific microorganisms directly from natural or clinical samples without the necessity of growing them in laboratory environment.
Sequencing of small RNA
Detection and quantitative marking of mRNA is of fundamental importance for the understanding of gene expression. The determining of the mRNA profile regulating the expression of other genes by the inhibition of mRNA translation and/or induction of mRNA degradation is of increasingly higher significance for molecular biology. It has been proved that mRNAs are key regulators in numerous biological processes, such as: development, differentiating, apoptosis and proliferation. Furthermore, mRNA profiles are to an ever greater extent regarded as biomarkers, particularly in terms of cancer research, due to which they may play a role as a new class of onkogenes or cancer suppressors. The next-generation sequencing with the Illumina platforms will thus give prospects for the discovery of molecular profiles associated e.g. with the given illness status.
RNA sequencing (RNA-seq)
Thanks to its speed, efficiency and accuracy, NGS sequenators offer a new alternative to classic RNA sequencing and transcriptome analysis. The RNA-seq method is an alternative for expression microarrays. On top of that, millions of readings during RNA sequencing can be compared to splicing spaces and thus enable the identification of RNA isoforms, discovery of new transcripts, fusion genes and non-coding RNA. The analysis can be performed for each point with reference genome. Finally, expressive data obtained from the NGS sequenator can be analysed with multiple tools used commonly in microarray analyses, including hierarchical clustering.
- colorectal cancer,
- breast cancer,
- prostate cancer,
- lung cancer,
- pancreas cancer,
- thyroid cancer,
- stomach cancer.
A) determination of genetic inclinations – analysis of genes contributing to the development of individual vulnerability to growth of cancer;
B) cancer treatment – sequencing of genes responsible for organism response to medicines.
Access Array™ (Fluidigm) is the first high-performance sample enrichment system intended to cooperate with all the most popular next-generation sequencing systems, including MiSeq. The Access Array system allows for the enrichment of samples and their marking for multiplex sequencing, as well as preparing libraries by tagging amplicons. Tagging of amplicons with the Access Array system reduces greatly the time of acquiring target sequences by combining the creation of amplicons with the library preparation process. It is a complete solution for demanding low-budget research project with a wide throughput range. The possibility of simultaneous analysing of 48 samples for 48 amplicons (48×48) sums up to 2304 reactions. The system also supports multiplexing which allows for gaining 480 amplicons per sample.
Scope of application:
- SNP analysis (Single Nucleotide Polymorphism)
- CNV analysis (Copy Number Variation)
- detection of mutation
- methylation analysis
- metagenome analysis
- analysis of single cells
Emulsion PCR is a new approach to molecular biology which aims at the detection of nucleic acids and quantitative product assessment. ddPCR offers an alternative method of absolute quantification and detection of rare allels compared to the conventional PCR method in real-time or qPCR. The ddPCR method is based on emulsion and droplet technology thanks to which a single sample is partitioned in real-time into 20,000 drops, enabling sensitive and precise detection of presence of even a single DNA particle in each of them. The system reads the target particles (positive) and all other (negative) particles, on basis of which the absolute number of target particles per sample is obtained without the need of referring to standard curves of reference gene (unlike in case of qPCR). This makes ddPCR a much more sensitive solution compared to the classical qPCR reaction.
Application of ddPCR:
- detection of rare mutations
- absolute quantification of gene expression, analysis of mRNA
- absolute quantification of the virus index, detection of pathogens, analysis of single cells
- detection of rare sequences
- absolute quantification of libraries for sequencing
- CNV analysis (Copy Number Variation)
Advantages of emulsion PCR:
- no need of referring to standard curves of reference gene
- higher precision may be achieved by increasing the repetition number
- high inhibitor tolerance
- analyses of complex mixtures
- results are generated in linear form depending on the number of copies which facilitates the analysing of small differences in fold change
We offer a wide range of biodata services in order to provide comprehensive and multi-dimensional analysis of the provided information.
We provide, among others:
- Data analyses with Next Generation Sequencing (NGS), such as: RNA-seq, smallRNA-seq, methylation analysis, sequencing analysis of amplicons, plasmides, de novo, microarray, metagenome, genetic panels etc.
- DNA and protein sequence analyses
- Filogenetic analyses
- Molecular modelling
- Protein-ligand interaction analyses
- Virtual Screening (VS)
- Analyses of binding of protein sequence, structure and function
- PDesigning and delivery of non-standard biological data analyses
- Tailored to the needs of specific data/research
- Assistance in the interpretation of biodata analyses delivered by the Customer