Next-Generation Sequencing

The introduction of next-generation sequencing (NGS) fundamentally changed how we approach scientific and diagnostic issues. Next-generation sequencing’s potential to produce large amounts of sequence data for a relatively low price led to a series of groundbreaking discoveries and ushered the field of genomics into a new era.

NGS Service

CeGaT has specialized in the use of NGS since its foundation. In addition to using it as a tool in our diagnostic services we offer this technology as a service for customer-specific projects.

Depending on the specific research questions and goals, our laboratory offers sequencing on the Illumina NovaSeq, HiSeq and MiSeq platforms.

Our services include:

  • Genome Sequencing

  • Targeted Sequencing (Exome and Panels: Agilent SureSelect; Amplicons)

  • Restriction site associated DNA Sequencing (RAD genotyping)

  • RNA Sequencing (Transcriptome Sequencing, smallRNA Sequencing)

  • Metagenome Sequencing

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Please do not hesitate to contact us – we are happy to design a custom concept for your project.

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Our team has a wide range of expertise in the processing of plant samples in addition to substantial experience with human and other mammalian samples.

Based on many years of using NGS technology, we have gained great expertise in high-throughput technology for processing hundreds of samples.
Our laboratories are equipped with five liquid handling workstation platforms from Agilent Bravo A/B and Hamilton STAR. Each of these platforms can be individually programmed allowing specific adaptations of our protocols based on your requirements.

The multiplexing strategy we have established in our laboratory allows the sequencing of thousands of samples in one sequencing run, or even in one lane and therefore represents true high-throughput sequencing.

We would be happy to demonstrate the feasibility of your high-throughput projects in a pilot study.

Our dedicated Customer Solutions team of scientists and bioinformaticians work with you closely to develop the best strategy for the realization of your projects.
After the preparation and sequencing of the samples we provide you with either the raw data for self-analysis or full analysis options.
Each project is led by a scientist and supervised by a project manager, who is your contact throughout the entire project.

NGS technology

The term next-generation sequencing (NGS) covers all innovative technologies for high-throughput sequencing. Next-generation sequencing is based on the parallel sequencing of millions of different DNA fragments in a single sequencing run. Compared with the traditional Sanger method this new technology allows an increase in throughput by several orders of magnitude and drastically lowers costs.

The steps from DNA to data can be divided into three sections: (1) Preparation of a DNA library, (2) Clonal, parallel amplification of DNA and (3) Sequencing.

Preparation of a DNA library

For the preparation of the DNA library, the DNA must first be fragmented. The ends of the DNA fragments are than repaired, as they are damaged by the fragmentation. The ligation of adapters to both sides of the repaired DNA fragments completes the preparation of the DNA library. These adapters contain sequence motifs that are required for subsequent steps (clonal amplification and the actual sequencing).

Clonal amplification

Illumina technology uses the so-called “Bridge-PCR” for clonal amplification of individual DNA fragments. This PCR is performed on the Flow Cell (a glass side similar to a microscope slide) on which the actual sequencing takes place. DNA fragments are linked via the adapters to the Flow Cell, then the Bridge-PCR is carried out. Oligos bound to the Flow Cell function as primers, and each individual DNA fragment forms a seperate cluster of identical DNA fragments.

Sequencing

Illumina’s Sequencing-by-Synthesis (SBS) is based on the Cyclic Reversible Termination (CRT) method. Each of the four nucleotides is linked with a different dye and modified by a terminator group. During a reaction cycle all four nucleotides are offered simultaneously to the polymerase for strand synthesis. After incorporation of a complementary nucleotide strand elongation is no longer possible due to the blocking effect of the terminator group. The four dyes of each nucleotide can be detected using Imaging: the dye and the terminator group are cleaved and a new synthesis cycle starts. The sequence of each cluster is thus simultaneously determined base by base.

Using sophisticated bioinformatics, individual base sequences (reads) are aligned to and compared with the reference genome for mutation detection.