Next Generation Sequencing (NGS)

The most thorough test method to provide a differential diagnosis for your patient

Generating a large amount of sequencing data in a massively parallel manner makes sequencing much faster and much more cost-effective, and thus allows us to sequence large panels, whole exomes (WES) and even whole human genomes (WGS).

 

  1. Next Generation Sequencing

Next Generation Sequencing (NGS)

Next Generation Sequencing describes a variety of high-throughput sequencing methods which highly increase the possible sequencing throughput and decrease the cost per sequenced base pair. In combination, this allows us to sequence not only single fragments or genes, as has been done by Sanger sequencing for many years, but also large panels, whole human exomes or even human genomes in a fast and affordable manner. 

CENTOGENE´s next generation sequencing department is mainly based on the Illumina sequencing technology, the current leading technology on the market. It is based on sequencing-by-synthesis (SBS), meaning that sequencing is achieved via synthesis of a new complementary DNA strand.

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How it works

There are several different approaches which all finally generate millions of small fragments of DNA which are tagged with further special sequences “patient bar codes” and adapters (so-called library) to enable them to bind on a sequencing chip (flow cell).

These chip-bound molecules are amplified (so-called bridge amplification) to generate clusters of identical copies in order to provide a sufficient signal (= clustering).

During the sequencing itself, nucleotides labeled with four different fluorescent dyes flow through the chip and are incorporated into a new DNA strand complementary to the original one. A terminator on each nucleotide ensures that only one nucleotide is incorporated. The sequencer optics (laser/camera) scan the chip to generate an image. Each cluster gives a signal depending on the incorporated nucleotide. By unblocking the last incorporated nucleotide and flowing of further nucleotides, the next cycle is initiated. In this manner, sequences (= reads) with up to 150 bp (from both directions of the DNA molecule) can be generated.

A bioinformatics pipeline is used to assemble the fragments by mapping the reads to the human reference sequence. Each base is sequenced several times thus providing highly accurate variant detection.

Taken together, this technology offers not only high accuracy, but also reliable performance in homopolymeric regions, which contributes to the high quality of CENTOGENE´s genetic diagnostics.

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