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Introduction to Next Generation Sequencing (NGS)

Introduction Nucleotides and Genome

All live on earth is based on the genetic code that is encrypted by the nucleic acids DNA and RNA. Nucleic acids consist of four different nucleotides (bases), A, C, G and T (U in RNA) and form specific base pairs in the double stranded DNA genome. The genetic information determined by the nucleic acid sequence is the basis for diagnostics, the development of new drugs, vaccines and treatment methods, and for NGS applications such as forensics and breeding of plants and animalsThe genome is the sum of all nucleic acids of an organism. The human genome contains about 3 billion base pairs.

First Generation Sequencing Opened the Era of Genomics

In 1977, the first DNA genome of the phage ΦX174 was sequenced by the Sanger sequencing method that is based on chain termination caused by the usage of specific modified nucleotides that inhibit DNA polymerisation during the polymerase chain reaction. Using the Sanger sequencing method, nucleotide fragments of up to 1000 bases in length can be sequenced. In order to sequence longer regions or genomes, initial cloning and PCR amplification steps are required prior to sequencing. 

Second Generation Sequencing - is the 1000$ Human Genome Realistic?

Roche454In 2005, Roche launched the first NGS technology, Roche 454. Following on from the Sanger method, NGS is often described as second generation sequencing technology. The 454 NGS technology is based on the fragmentation of the DNA segment or genome of interest into small sections of 100-1000 nucleotides in length prior sequencing. By application of a method called pyrosequencing of these small DNA fragments (indicated as reads), NGS technology enables the parallel sequencing of millions of short fragments which can subsequently be assembled using specific algorithm to generate a consensus sequence of the sequenced target DNA. The Next generation sequencing technology enables the sequencing of a whole human genome in one run. The short fragments are subsequently assembled using bioinformatics software tools into the original consensus sequence of the complete genome. Today, NGS platforms from different providers are on the market, including Illumina, Ion Torrent, and Roche.

Existing technologies are constantly improving, and new technologies appear on the market whereas older sequencing platforms are retired due to high operational costs or outdated technology. More detailed information about the various technologies is available on the NGS Technologies page. The clear market leader is Illumina®, followed by Ion Torrent, Roche, PacBio and others, including Qiagen and Oxford Nanopore TechnologiesmiseqSecond generation sequencing enables a read length of up to 1000 nucleotides, depending on the specific technology, protocol and chemistry applied. The read number per sequencing run varies from 1 million reads up to a maximum of 3 billion reads obtained using the Illumina® HiSeq platform.

The sequencing of the first human genome using the Sanger sequencing method took more than one decade and cost approximately three billion dollars. The recently launched complete automated ultra high-throughput sequencing workflow from Revolocity™ allows the parallel sequencing of about 120 human genomes in one run, taking eight days from sample to assembled genome. Revolocity™ technology aims for a sequencing rate of 30,000 human genomes per year, corresponding to a 400,000 times faster and about three million times cheaper sequencing process for one human genome compared to the first human genome sequenced by the Sanger method. This calculation does not take into consideration that the sequencing of the first human genome required the involvement of hundreds of scientists, whereas this new technology is fully automated.

On March 3. 2016, Veritas Genetics introduced the Veritas myGenome, termed as the worlds first 999$ whole human genome sequencing service including data interpretation and genetic counseling. This offer was made for participants of the Personal Genome Project (PGP). 

Big Data: the Challenge of Bioinformatics and Data Storage in NGS

One of the greatest challenges in NGS is the correct assembly of the short reads to the whole genome, revolocityespecially in regions with structural variants (SV) that contain insertions, deletions, or poly-repetitive sequences. Numerous software tools have been developed to address this issue. Another issue is the sequence data analysis. Numerous software tools are available such as for quality control & data trimming, assembling & binning, alignment & mapping, data analysis, variant calling, data base search or visualization. For data analysis and storage huge computing power and storage space is needed. This is often realized by cloud computing

In order to use sequencing technology most beneficial, the sequence information should be stored in data bases with public access. There are many different data bases for human genetics, cancer genetics, animal genetics, plant genetics or microbial genetics

Learn more about bioinformatics in next generation sequencing

Third Generation Sequencing - Enables Real Time Sequencing

The so called third generation sequencing technologies (e. g. SMRT PacBio and Oxford Nanopore Technologies) enable real time sequencing of long reads. These technologies have specific advantages in that they allow sequencing in real time and do not require amplification of the target sequence prior to sequencing. MinIonAverage sequencing read length is currently about 10k nucleotides depending on the technology. Theoretically, sequencing reads can be as long as the DNA fragment used for sequencing. The number of sequenced reads per run is lower compared to second generation sequencing technologies. One major advantage of this technology is its ability to sequence complicated SV regions. The Oxford Nanopore Technologies platform, in particular, permits simple, fast, and cost-effective sequencing of long DNA fragments, although its inherent drawback is a high error rate. As the technology is new on the market, however, improvements are expected in the near future.

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