Next Generation Sequencing

-Lacatus Radu-

Introduction
In 2000 a couple of very famous papers were published by competing groups describing for the first time the complete human genome. That was done using first generation sequencing. Back then they used millions of clones and after 9 months, funded with billions of dollars they were able to sequence the entire human genome. Today you can achieve the same goal with billions of much shorter reads in a week for thousands of dollars. This can be achieved with Next generation sequencing.

The method
The experiment starts with millions of copies of the DNA that you are interested in measuring. The DNA is then randomly fragmented. The pieces resulted from this process arent perfect, so they go through a process that turns them into the so called reads. The process consists of blunting, also called end repair, a-tailing, ligating with adapters. A collection of processed fragments is called a library. The location information is now lost but you have billions of reads with the length usually ranging from 35 to 100-150 base pairs. Mapping the reads to their correct place in the initial DNA sequence is possible due to the large amount of data. Once you map the entire genome, the first useful experiment that comes to mind is variant detection. In other words, the question is do humans change on a specific location in the genome or is everybody the same? This technology facilitates finding the answer to this question. For example, if in a specific location, for the same person there are different sequences that have two different bases, the conclusion would be that its a heterozygous gene that varies and that person had parents with different genes in that location. The large amount of data also makes it easy to spot the errors generated by the technological process.

Next-generation methods
MPSS (Massively parallel signature sequencing) was the first of the next generation sequencing
methods. The MPSS method was bead-based and used a complex approach of reading the sequence in increments of four nucleotides for adapter ligation and adapter decoding. This made the method susceptible to sequence-specific bias or loss of specific sequences. The MPSS method was rendered obsolete around 2004 by the development of sequencing-by-synthesis.

Polony sequencing was among the first next-generation sequencing systems and was used to
sequence a full genome in 2005.

454 pyrosequencing was developed by 454 Life Sciences and is similar to a parallel version of

pyro sequencing. A clonal colony is formed in an oil solution by water droplets containing a DNA template. The sequencing machine contains many picoliter-volume wells each containing a single

bead and sequencing enzymes. Pyrosequencing generates light detecting the individual nucleotides added to the DNA by luciferase, and the resulting data is used to generate sequence read-outs.

Illumina , a technology originally developed by Solexa, brought significant improvements to the

basic technology around 2010 when the HiSeq 2000 was released. The simplicity of sample preparation went hand in hand with the ease of method development and made this technology the most widely used NGS instrument around the world. Ion semiconductor sequencing is a system based on using standard sequencing chemistry, but with a novel, semiconductor based detection system. The newer Ion Torrent system uses a very similar approach to the original 454 pyroseqeuncing. A semiconductor chip in which PCR beads are loaded detects the change in PH with an ion sensor located at the bottom of the well. The data is then read out in flowgram format.