ngs  

Mar 29, 2015 • Michael Chen

In the old days, genome sequencing was expensive and slow. For example, human genome project took more than 10 years, work parallelly in several countries, and cost billions of dollars. Traditionally, genome sequencing was based on Sanger’s chain termination method. On 2005, first next generation sequencing (NGS) platform became available. In the following years, several NGS platform hit the market. The cost for sequencing dropped sharply. Personal genome became possible as well.

There are several NGS platforms invented by different companies. Some examples are Ilumina/Solexa, Roche 454, and Applied Biosystems. Their technique details are different, but they share similar workflow. The major workflow are:

Second generation sequencing

Let’s take the platform of Ilumina/Solexa as a example to see the workflow. The NGS platforms of Ilumina/Solexa are popular among scientific communities.

Before the process, DNA or cDNA (from RNA) samples should be prepared. These samples were cut to shorter nucleotide sequences. In Ilumina/Solexa platform, the length of each sequence is about 300 base pairs. Then, these short nucleotide sequences attach to special glass solid plate, where bridge amplifications are performed. After the process, original sample grows into clusters of millions of repeated sequences.

solid phase amplification

Ilumina/Solexa adapts four-color cyclic reversible termination to do sequencing. In each cycle, complementary special-made necleotides attach to the template, and a single sequence is record. After several cycles, the sequencer gets millions of reads. Reads mean these short sequences got by NGS platform.

four-color cyclic reversible termination

After the sequencer gets these reads. We have to assemble these reads into original sequence. If the species of the sample was sequenced before, we may utilize the sequence as reference genome to do assembly. Otherwise, de novo assembly is needed.

resequencing with reference genome

NGS is widely utilized among scietific communities. There are many NGS applications, some of them are:

With the fall of the cost of NGS sequencing, personal genomics become possible and feasible. Maybe one day, genome data and other personal profile data will cooperate into electronic health record and become standard medical practice.