Ośrodek Genomiki

Dear Colleagues, Dear Guests,

We would like to present you our newly established Genomic Centre, a unit providing services in the area of genetic variation analysis.

We encourage you to review our offer:

To obtain more information about our services, request a quote or place an order, please contact us:

Prof. Wojciech Branicki

E–mail: wojciech.branicki@uj.edu.pl

Tel.: +48 12 664 61 11

Next-generation sequencing

Next-generation sequencing (NGS), also known as massively parallel or high-throughput DNA sequencing, is a term describing novel DNA sequencing technologies which have revolutionized genomic research in the last decades. Because its unprecedented speed and throughput allow us to study DNA variation on a much larger scale than ever before, NGS became an indispensable tool helping to address complex biological problems. Its role, however, is not confined to basic scientific applications any longer, for the importance of deep sequencing increases also in the fields of clinical diagnostics or forensics, ensuring thus a constant development of the technology and a growing demand for NGS services.

NGS at MCB

As a state-of-the-art life science research facility, Malopolska Centre of Biotechnology is inherently well suited to provide both academic and non-academic entities with the opportunity to exploit the benefits of the NGS technology according to their needs. Therefore, Genomics Centre at MCB – a modernly equipped unit, staffed with experienced technicians and scientists dedicated to genome variation studies - is now offering its services in the area of next-generation sequencing to all interested researchers and institutions. We believe that our expertise combined with befitting infrastructure will let us assure the best quality of rendered services and the output data.

At present, we carry out high-throughput DNA sequencing experiments utilizing two Ion Torrent platforms - PGM and Proton. We chose this semiconductor-based technology, because it allows translation of chemically encoded information – nucleobases A, C, G, T - to digital information in a direct manner (no modified nucleotides or optics are involved), hence sequencing is done faster and more cost-effectively than in any other available NGS technology. The Ion Torrent supports numerous NGS applications, including the most common, like whole transcriptome sequencing, targeted DNA/RNA sequencing, and many more.

Detailed NGS offer

Whole transcriptome sequencing (RNA-Seq)

Used to analyze the sequence of all coding and non-coding RNAs present in a sample at a given time after removal of ribosomal RNA or poly(A) selection, it enables also the determination of relative abundance of identified RNA molecules and therefore is a perfect approach for both discovery and gene expression analysis. Unlike gene expression microarrays, RNA-Seq does not require species- or transcript-specific probes, hence it allows detection of novel transcripts, gene fusions, single nucleotide variants, indels and splice variants. Moreover, it delivers increased specificity and sensitivity, while maintaining the strand orientation, which may aid for instance in understanding of transcriptional regulation.

Input requirements: 100 ng - 1 μg of high quality total RNA (ideally RNA integrity number >7). Libraries are constructed from 10-15 ng of poly(A) RNA or 25 ng of rRNA-depleted RNA following the total RNA enrichment.

We accept also different types of biological samples (i.e. cell suspensions, tissues) for an in-house RNA extraction (for additional cost).

Targeted human transcriptome sequencing for gene expression analysis

Perfect alternative to gene expression microarrays for fast and cost-effective human transcriptome sequencing. The Ion AmpliSeq Transcriptome Human Gene Expression Panel, covering >95% of human RefSeq genes (nearly 21,000 RNA targets), provides an attractive solution for researchers interested in gene-level quantification and differential gene expression analysis. The small amplicon designs enable the use of the panel with RNA isolated from fixed tissues, such as formalin fixed paraffin embedded (FFPE) samples and do not require poly(A) selection or rRNA depletion.

Input requirements: 50-100 ng of total RNA (preferably DNase-treated)

We accept also different types of biological samples (i.e. cell suspensions, tissues) for an in-house RNA extraction (for additional cost).

Small RNA sequencing

A technique used to isolate and sequence small RNA species, such as microRNA (miRNA), short-interfering RNA (siRNA), piwi-interacting RNA (piRNA) and others, with high sensitivity and dynamic range. It enables the discovery of novel sequences and analysis of differential expression of all small RNAs present in a sample. Small RNA sequencing provides a useful tool in revealing the role of non-coding RNA, studying post-transcriptional regulation of gene expression or identifying new biomarkers.

Input requirements: 300 ng - 1 μg of high quality total RNA (RNA integrity number >6) containing the small RNA fraction (10-40 nt). We recommend using mirVana miRNA Isolation Kit for RNA isolation.

We accept also different types of biological samples (i.e. cell suspensions, tissues) for an in-house RNA extraction (for additional cost).

Targeted RNA sequencing (ready-to-use or custom panels)

Often employed instead of whole transcriptome sequencing, whenever it is useful to get a focused view only on a particular set of genes or regions, e.g. implicated in a given disease. Targeted RNA sequencing with Ion AmpliSeq RNA panels offers the flexibility to either select one of the ready-to-use solutions:

  • Ion AmpliSeq RNA Cancer Panel targeting 50 oncogenes and tumor suppressor genes including the KRAS, BRAF, and EGFR genes,
  • Ion AmpliSeq RNA Fusion Lung Cancer Research Panel targeting fusion transcripts included in the ALK, RET, ROS1, and NTRK genes,
  • Ion AmpliSeq RNA Apoptosis Panel targeting genes associated with death receptor–mediated apoptosis, c-myc-mediated apoptosis, and p53-mediated apoptosis,

or create your own design that best suits your needs. Ion AmpliSeq RNA Custom Panels enable scientists to choose from more than 20,000 genes to survey gene expression fold changes. Simply determine your targets of interest and we will take care of the panel design or use a free online tool – Ion AmpliSeq Designer to do it on your own.

Input requirements: 500 pg of high quality RNA or 5 ng FFPE RNA per primer pool

We accept also different types of biological samples (i.e. cell suspensions, tissues) for an in-house RNA extraction (for additional cost).

Exome sequencing

An approach restricted to sequencing of the protein-coding regions of genome, which is most frequently exploited by genetic researchers trying to identify genes implicated in rare or complex disorders, but also gets more and more often applied by clinicians for diagnostic purposes. It enables characterization of single nucleotide variants, copy number variations, small insertions or deletions and de novo mutations in the regions most likely to affect the phenotype. Our workflow is based on ultra-high multiplex PCR exome enrichment, which typically covers >97% of protein-coding sequences and yields >90% of on-target bases. Paired with Ion Proton sequencing, it provides an affordable solution with a short turnaround time.

Input requirements: 50-100 ng of genomic DNA

We accept also different types of biological samples (i.e. blood, tissues) for an in-house DNA extraction (for additional cost).

Targeted DNA sequencing (ready-to-use or custom panels)

Designed to match the requirements of any targeted sequencing research application, Ion AmpliSeq offers a broad range of ready-to-use panels as well as fully customizable panels. Regardless of the sample type (gDNA, cfDNA, FFPE) or species you work with, it provides a powerful tool for detection of various mutation types within your regions of interest. For the details concerning ready-to-use panels developed specifically for cancer research, inherited and infectious diseases, human identification, and more, please visit www.ampliseq.com. To give some examples:

  • Ion AmpliSeq™ Inherited Cancer Research Panel
  • Ion AmpliSeq™ Comprehensive Ovarian Cancer Research Panel
  • Ion AmpliSeq™ BRCA1 and BRCA2 Panel
  • Ion AmpliSeq™ Cancer Hotspot Panel v2
  • Ion AmpliSeq™ Comprehensive Cancer Panel
  • Ion AmpliSeq™ Inherited Disease Panel
  • HID-Ion AmpliSeq™ Identity Panel

To create your own custom panel, use the Ion AmpliSeq Designer – a free online tool, or supply us with the list of your targets and we will do it for you.

Input requirements: 1-10 ng of DNA per primer pool

We accept also different types of biological samples (i.e. blood, tissues) for an in-house DNA extraction (for additional cost).

Other

The above-mentioned NGS services belong to the most common and offer a satisfactory choice for majority of the researchers. However, if you do not find an approach suitable for your research problem among them, please feel free to contact us, tell us about your project and we will do our best to find a solution that will be tailored to your needs.

Capillary electrophoresis

Capillary electrophoresis (CE) is a method commonly used for DNA analysis, as it allows rapid electrophoretic separations for various applications. The MCB‘s Genomics Centre offers CE services utilizing the 3500 Series Genetic Analyzer manufactured by Applied Biosystems. The instrument can be used for examination of products of Sanger sequencing and DNA fragment analysis, which is applied in a number of genetic assays (e.g. microsatellite genotyping, MLPA, AFLP, LOH). The most frequent implementations of our Genetic Analyzer include the following:

Sanger sequencing

The DNA sequencing method developed by Frederick Sanger has been for many years the most widely used DNA sequencing technique. Proven through the decades of use to generate highly accurate and reliable data, it is recognized as the gold-standard in genome variation analyses. Nowadays, despite the development of the next-generation DNA sequencing technologies, Sanger sequencing still remains in demand, especially for smaller-scale projects, long read sequencing and NGS results validation.

Microsatellite (STR) analysis

Microsatellites (or short tandem repeats) present a large selection of highly informative markers, therefore their analysis is a particularly useful tool in genetic linkage and association studies, but is also often employed in identifying individual organisms (e.g. human identification). The assay includes PCR amplification of the microsatellite loci using fluorescently labelled primers. Resulting PCR products are then analysed by capillary electrophoresis to separate the alleles by size.

SNP-minisequencing (SNaPshot Multiplex System)

Minisequencing is a common method with applications not only in genomic research, but also in forensics, especially useful for degraded or low copy number DNA analysis. SNaPshot Multiplex System allows investigation of up to 30 SNP markers simultaneously by using single-base extension technique. In principle, user-defined primer is extended with the dye-labelled ddNTP, the products are then separated through capillary electrophoresis and after fluorescence detection, alleles of a single marker can be identified. Sensitive allele frequency detection (5%) and multiplexing capability makes SNaPshot a perfect solution for SNP validation.