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Next generation sequencing

First generation sequencing (also known as Sanger or population sequencing) is based on the analysis of a mixture of DNA molecules, resulting in a consensus sequence. This method is not suitable to identify minority variants in complex populations.

Next generation sequencing (also known as deep sequencing or massive parallel sequencing) has solved this problem, thereby greatly improving the resolution of sequencing. Minority species can now be effectively detected, which is crucial to analyse DNA from complex populations such as viral infections.

DDL has chosen at this moment, to use the Illumina technology, which has a very low error rate and yields highly reliable data.

Next generation sequencing can be applied on various DNA sources, e.g., PCR amplicons, total RNA, or total genomic DNA.

DDL has developed several dedicated data analysis pipelines for data analysis, variant calling and tailor-made reporting, e.g., for HCV, HBV and influenza virus. For more information see bio-informatics.

Amplicon-based sequencing
PCR-generated ampicons of any size can be directly sequenced by NGS.

Total RNA sequencing
Certain viral RNA targets are highly heterogeneous and it is not always possible to use specific PCR primers for target amplification. As an alternative approach, it is possible to perform sequence-independent amplification and total RNA deep sequencing. This provides sequence information on all RNA molecules in a sample, including the target of interest.

Whole genome sequencing
Total genomic DNA, e.g., from cultured bacteria, can be analyzed by next generation sequencing to analyse genome composition, perform multilocus sequence typing or detect mutations and polymorphisms.

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