Bioinformatics Genomics Next Generation Sequencing

Methods of NGS

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Roche 454 System

Roche 454 was the first commercially successful next generation system. This sequencer uses pyrosequencing technology. Instead of using dideoxynucleotides to terminate the chain amplification, pyrosequencing technology relies on the detection of pyrophosphate released during nucleotide incorporation. The library DNAs with 454-specific adaptors are denatured into single strands and captured by amplification beads followed by emulsion PCR. Then on a picotiter plate, one of dNTP (dATP, dGTP, dCTP, dTTP) will complement to the bases of the template strand with the help of ATP sulfurylase, luciferase, luciferin, DNA polymerase, and adenosine 5′ phosphosulfate (APS) and release pyrophosphate (PPi) which equals the amount of incorporated nucleotide. The ATP transformed from PPi drives the luciferin into oxyluciferin and generates visible light. At the same time, the unmatched bases are degraded by apyrase. Then another dNTP is added into the reaction system and the pyrosequencing reaction is repeated.

The read length of Roche 454 was initially 100–150 bp in 2005, 200000+ reads, and could output 20 Mb per run. In 2008 454 GS FLX Titanium system was launched; through upgrading, its read length could reach 700 bp with accuracy 99.9% after filter and output 0.7 G data per run within 24 hours. In late 2009 Roche combined the GS Junior, a bench top system into the 454 sequencing system which simplified the library preparation and data processing, and output was also upgraded to 14 G per run. The most outstanding advantage of Roche is its speed: it takes only 10 hours from sequencing start till completion. The read length is also a distinguished character compared with other NGS systems.


(Sequencing by Oligo Ligation Detection) SOLiD was purchased by Applied Biosystems in 2006. The sequencer adopts the technology of two-base sequencing based on ligation sequencing. On a SOLiD flow cell, the libraries can be sequenced by 8 base-probe ligation which contains a ligation site (the first base), cleavage site (the fifth base), and 4 different fluorescent dyes (linked to the last base). The fluorescent signal will be recorded during the probes complementary to the template strand and vanished by the cleavage of probes’ last 3 bases. And the sequence of the fragments can be deduced after 5 rounds of sequencing using ladder primer sets.

The read length of SOLiD was initially 35 bp reads and the output was 3 G data per run. Owing to the two-base sequencing method, SOLiD could reach a high accuracy of 99.85% after filtering. At the end of 2007, ABI released the first SOLiD system. In late 2010, the SOLiD 5500xl sequencing system was released. 

Illumnia GA/HiSeq System

In 2006, Solexa released the Genome Analyzer (GA), and in 2007 the company was purchased by Illumina. The sequencer adopts the technology of sequencing by synthesis (SBS). The library with fixed adaptors is denatured to single strands and grafted to the flow cell, followed by bridge amplification to form clusters which contain clonal DNA fragments. Before sequencing, the library splices into single strands with the help of linearization enzyme, and then four kinds of nucleotides (ddATP, ddGTP, ddCTP, ddTTP) which contain different cleavable fluorescent dye and a removable blocking group would complement the template one base at a time, and the signal could be captured by a (charge-coupled device) CCD.

Compact PGM Sequencers

Ion Personal Genome Machine (PGM) and MiSeq were launched by Ion Torrent and Illumina. They are both small in size and feature fast turnover rates but limited data throughput. They are targeted to clinical applications and small labs.

Ion PGM from Ion Torrent

Ion PGM was released by Ion Torrent at the end of 2010. PGM uses semiconductor sequencing technology. When a nucleotide is incorporated into the DNA molecules by the polymerase, a proton is released. By detecting the change in pH, PGM recognized whether the nucleotide is added or not. Each time the chip was flooded with one nucleotide after another, if it is not the correct nucleotide, no voltage will be found; if there are 2 nucleotides added, there is double voltage detected. PGM is the first commercial sequencing machine that does not require fluorescence and camera scanning, resulting in higher speed, lower cost, and smaller instrument size. Currently, it enables 200 bp reads in 2 hours and the sample preparation time is less than 6 hours for 8 samples in parallel.

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