In the previous post, we saw the impetus behind the technological innovation that led to development of next generation sequencing. So, for aficionados, Dr Sandeep Pingle, Roundtable blog editor will continue with his series and discuss in brief, the newer technologies that are grouped under the moniker of next generation sequencing.
Development of next generation sequencing was spurred by the need for rapid, low-cost, high fidelity genomic analyses that was unlikely to be provided by the first generation Sanger sequencing. This first-generation capillary-based sequencing involved synthesizing a complementary DNA template using natural 2′-deoxynucleotides (dNTPs) followed by termination of synthesis using fluorescent or radiolabeled 2′,3′-dideoxynucleotides (ddNTPs). This label corresponded to the nucleotide identity of its terminal position. The DNA sequence was then read by high-resolution electrophoretic separation of the products in a capillary-based gel.
In contrast, next generation sequencing is a high-throughput sequencing technology where the sequential addition of nucleotides to immobilized, arrayed DNA templates is monitored and later interrogated to reveal their sequences. Compared to the Sanger method, this technology can sequence billions of sequences in parallel. It uses one of these techniques:
- Reversible Chain Terminator
- Ligation-based Sequencing
Imaging techniques such as bioluminescence measures and four-color imaging are used following which the sequencing reads are aligned to a known reference sequence. In some cases, these reads are assembled de novo. This is a rapid, inexpensive, high efficiency and more accurate technique compared to Sanger sequencing.
A key feature of next generation sequencing is its digital nature, compared to the analogue first-generation technology . This makes it possible to read the same stretch of DNA sequence multiple times to improve the signal-to-noise ratio (termed over-sampling). This is a highly desirable feature for clinical sequencing (for example, in case of cancer tissue) to reliably detect mutations, rearrangements and other genomic alterations. The amount of over-sampling used during sequencing is described as the depth of coverage. For whole-genome sequencing, an average depth of 30-fold is considered necessary to reliably detect nucleotide alterations. In contrast to whole-genome sequencing, targeted sequencing strategies are frequently employed by sequencing only the coding exons of the gene (exome sequencing) or the transcriptome (RNA-sequencing). These strategies require higher coverage to reliably detect abnormalities. However, the advantage of targeted sequencing using next generation sequencing technologies is the relatively lower cost for much higher sequence coverage depth.
The notable commercial platforms for next generation sequencing and the technologies they use are :
- Illumina/Solexa’s GA – Reversible Chain Terminator
- Roche/454’s GS FLX Titanium – Pyrosequencing
- Life Technologies/APG’s SOLiD 3 – Ligation-based Sequencing
- Helicos Biosciences HeliScope – Reversible Chain Terminator
- Polonator G.007 – Ligation-based Sequencing
- Pacific Biosciences – Real-Time
In addition, other companies including IBM, Oxford Nanopore Technologies, Intelligent Bio-Systems, LaserGen, and NABsys are developing next generation sequencing technologies.
Owing to the tremendous technological advancements, scientists and physicians are increasingly sequencing genomes, exomes or transcriptomes, which have direct applications in basic research and the clinic.
- Sandeep Pingle is the San Diego Editor for the blog “Roundtable Review” by Oxbridge Biotech Roundtable
- Meyerson M. et al (2010) Advances in understanding cancer genomes through second-generation sequencing. Nature Reviews Genetics. PMID: 20847746.
- Metzker ML. (2010) Sequencing Technologies – The Next Generation. Nature Reviews Genetics. PMID: 19997069