15.13 : Sequenciamento de Nova Geração

Next-generation sequencing is the collective name for a group of technologies that can facilitate the relatively rapid generation of sequence data from many different species, or from groups of individuals.

These data can be used for fine-scale population genetics or clinical studies, and these technologies are faster and more cost-efficient than traditional sequencing methods.

There are many Next-Generation sequencing methods, but arguably the most popular is reversible terminator sequencing. Others include pyrosequencing and sequencing by ligation.

In reversible terminator sequencing - pure genomic DNA is first cut into smaller fragments of 100-1000 base pairs. Special adapters- which are oligonucleotide sequences with primer sites - are then ligated to both ends of these DNA fragments.

The single-stranded DNA fragments are then loaded on a specialized chip called a flow cell that is pre-coated with short oligonucleotide sequences complementary to the adapter sequences. The DNA fragments bind to the chip through their adapters.

In the amplification stage, DNA polymerase is then used to synthesize a complementary DNA strand. The double-stranded DNA is denatured, and the original template is washed off.

The remaining complementary strand then bends over and forms a bridge-like structure using the second adapter on the other end.

DNA polymerase can then synthesize a complementary strand of the bent DNA strand - a process called bridge amplification.

The double-stranded bridge is again denatured to generate linear, single-stranded DNA, which can subsequently make new bridges on the chip.

The DNA polymerase continues these cycles of bridge amplifications, synthesizing thousands of copies of a DNA fragment and resulting in the generation of clonal clusters.

All reverse complementary strands are then washed off the chip, leaving only the forward DNA strands immobilized on the chip.

The sequencing reaction begins with DNA Polymerase adding modified dNTPs tagged with four different fluorescent labels. These dNTPs also contain a 3’ blocking moiety, called a reversible terminator, that only allows for the addition of one dNTP at a time.

After each round of dNTP addition to the growing strand, the chip is imaged, and the emission wavelength from each DNA cluster is used to identify the base.

The sequence of fluorescence images of the flow cell captured after each dNTP addition is used to determine the sequence of the DNA fragments.