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Tentative paragraphs:

(Dallorto Eleonora)

1. Illumina sequencing

Introduction to NGS

Next generation sequencing (NGS), massively parallel or deep sequencing are related terms that describe a DNA sequencing technology . Using NGS an entire human genome can be sequenced within a single day. In contrast, the previous Sanger sequencing technology, used to decipher the human genome, required over a decade to deliver the final draft. The short read, massively parallel sequencing technique was a fundamentally different approach that revolutionized sequencing launching the “next generation” in genomic science.

Illumina® sequencing technology was developed by Shankar Balasubramanian and David Klenerman of Cambridge University, later founder of Solexa, a company acquired by Illumina®. (Go for a brief history of Illumina® sequencing https://emea.illumina.com/science/technology/next-generation-sequencing/illumina-sequencing-history.html?langsel=/it/)

Illumina® sequencing is based on the incorporation of reversible dye terminators that enable the identification of single bases as they are incorporated into DNA strands.

This technique offers some advantages over traditional sequencing methods such as Sanger sequencing. The automated nature of Illumina® sequencing makes it possible to sequence multiple strands at once and obtain actual sequencing data quickly. The innovative and flexible sequencing system enables a broad array of applications in genomics, transcriptomics, and epigenomics.

The Basics of NGS Chemistry

A DNA polymerase catalyses the incorporation of fluorescently labelled deoxyribonucleotide triphosphates (dNTPs) into a DNA template strand during sequential cycles of DNA synthesis. During each cycle, at the point of incorporation, the nucleotides are identified by fluorophore excitation. The critical difference is that, instead of sequencing a single DNA fragment, NGS extends this process across millions of fragments in a massively parallel manner.

Illumina® sequencing technology works in four basic steps:

• Library Preparation: The first step is to break up the DNA into more manageable fragments of around 200 to 600 base pairs. Short sequences of DNA called adaptors are attached to the DNA fragments. Alternatively, “tagmentation” combines the fragmentation and ligation reactions into a single step: enzymes called transposases randomly cut the DNA into short segments ("tags"), which greatly increases the efficiency of the library preparation process. Adapter-ligated fragments are then PCR amplified and gel purified.
• Cluster Generation: The library is loaded into a flow cell where fragments are captured on a lawn of surface-bound oligos complementary to the library adapters. The complementary DNA binds to primers on the surface of the flowcell and DNA that does not attach is washed away. Unlabeled nucleotide bases and DNA polymerase are then added to lengthen and join the strands of DNA attached to the flowcell. This creates “bridges” of double-stranded DNA between the primers on the flowcell surface. The double-stranded DNA is then broken down into single-stranded DNA using heat, leaving several million dense clusters of identical DNA sequences. When cluster generation is complete, the templates are ready for sequencing.
• Sequencing: Illumina® uses a proprietary reversible terminator based method that detects single bases as they are incorporated into DNA template strands. Primers and fluorescently labeled terminators (terminators are a version of nucleotide base (A, C, G or T) that stop DNA synthesis) are added to the flow cell. The primer attaches to the DNA being sequenced. The DNA polymerase then binds to the primer and adds the first fluorescently labelled terminator to the new DNA strand. Once a base has been added no more bases can be added to the strand of DNA until the terminator base is cut from the DNA. Each of the terminator bases (A, C, G, and T) give off a different colour. The fluorescently labelled terminator group is then removed from the first base and the next fluorescently base can be beside added. And so, the process continues until millions of clusters have been sequenced.
• Data Analysis: During data analysis and alignment, the newly identified sequence reads are aligned to a reference genome, this looks for matches or changes in the sequenced DNA. Following alignment, many variations of analysis are possible: DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions (clusters of genes), full chromosomes, or entire genomes.

For a detailed animation of Illumina® sequencing technology click and watch this video --> 

2. Paired-end sequencing

3. Ion Torrent sequencing

4. Roche sequencing

5. Pacific Bio and Nanopore long-run sequencing