Sequencing: Sanger and Next generation sequencing (NGS)
When we find a pathogenic mutation our technicians confirm the result by analysing a reference sample. When a pathogenic mutation is found by Next Generation Sequencing, it is always confirmed by Sanger sequencing.
SANGER sequencing is based on the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators. This is still the method of choice for most genes and small panels.
Next Generation Sequencing (NGS) is a revolutionary technology that delivers fast, inexpensive and accurate genetic information. Next generation sequencing methods allow the parallel sequencing of multiple genes and facilitate the clinical diagnostic steps in complicated phenotypes.
We are pleased to provide details on how Next Generation Sequencing is performed at Centogene
(see pdf).
NGS sequences in parallel short, randomly overlapping fragments of DNA. The sequence information then assembled based on either a known reference sequence or assembled de novo; variants are detected with nearly 100% sensitivity. An amplicon library for high-throughput parallel sequencing is prepared by Polymerase Chain Reaction (PCR) generating amplicons containing different adaptors at the 5’-prime and 3’-prime ends. In addition to the entire coding sequence, amplicons cover at least 20 base pairs of 5’- and 3’-flanking regions of the corresponding introns. After purification of PCR amplicons with Agencourt AMPure beads according to the manufacturers protocol (Beckman Coulter), individual PCR products are quantified using the Quant-iT PicoGreen dsDNA reagent (Molecular Probes, Invitrogen). An equimolar amplicon pool is generated and prepared for the subsequent emulsion PCR step. The amplification reaction, breaking of the emulsion, enrichment of beads carrying amplified DNA and the subsequent pyrosequencing is performed using the workflow for amplicon Sequencing as recommended by the manufacturer (454/Roche Diagnostics). Next Generation Sequencing is conducted on the Genome Sequencer Junior Titanium System (454/Roche Applied Science). All data is analyzed with the GS Amplicon Variant Software (Roche Diagnostics). Selected amplicon size ranges from 200-500 bp which enables bi-directonal pyrosequencing and maximum quality reads resulting in the highest confidence calls for low frequency variants. In addition, the sensitivity of variant detection is directly linked to the fold-coverage of the region (Amplicon) for example a 40x coverage will result in greater than 99.9% chance of detecting a heterozygote. High quality variant detection is assured by obtaining at least 50 independent forward and reverse reads per amplicon (bi-directional sequencing with at least 50x coverage).
