Evolution of DNA Sequencing Technologies
Knowledge of DNA sequence is indispensable for the basic biological research, medical diagnostic, biotechnology, forensic, virology, and biological systematics. Methods to decipher sequence have tremendously evolved since the last four decades. We have come a long way from running big gels and manually interpreting DNA sequence to the NGS era with highest throughput platforms that can sequence whole genome in just a few hours. First method used for DNA sequencing was the chain-termination method developed by Frederick Sanger in 1975 followed by chemical-sequencing method developed by Maxam and Gilbert in 1977. Automation of Sanger sequencing was revolutionary. The first automated DNA sequencer was launched in 1987. The main limitation was its low throughput, high cost, and long time to sequence.
Next or second-generation sequencing (SGS) platforms (The Roch, SOLiD, Illumina) were introduced to improve the throughput. In SGS, thousands of DNA fragments are sequenced in parallel to achieve high throughput that generates huge amount of sequence data in a single run. However time taken in this process is longer due to huge number of scanning and washing cycles. Dephasing and PCR amplification are the major drawbacks that add to the complexity of the procedure, and also results in increase errors and time for sequencing. The short reads produced are difficult to align and assemble giving challenge to the bioinformaticians. To further improve the time and cost, Ion Torrent sequencer was introduced. It is based on sequencing by synthesis (SBS) chemistry that does not require light, scanning, and cameras to monitor the base incorporation. However, like other SGS technologies, this technology also involves PCR amplification and washing and scanning cycles.
Third-generation sequencing (TGS) was developed to increase the rate of sequencing, simplify the sample preparation, increase sequencing throughput, and to ultimately decrease cost of sequencing. This technology is based on single-molecule sequencing, without the need for halting between the base incorporation.
The first commercially available TGS instrument was Helicos Genetic Analysis Platform that involves fixing-single DNA molecules on the glass-flow cells, and extending these molecules using primers and fluorescently labelled nucleotide analogues called virtual terminator nucleotides. This technology has advantage over SGS technologies as it does not require PCR amplification. However, time to sequence single nucleotide is long as this technology too requires halting, and read length is low. Unlike SGS, there is no issue of dephasing and many hundreds of millions of sequencing reactions can be carried out asynchronously, a hallmark of TGS. Not only DNA but RNA can also be sequenced by just replacing the DNA polymerase to reverse transcriptase enzyme, erasing the need for RNA to cDNA conversion. However, this technology could not overcome all the shortcomings of SGS.
The single-molecule real-time (SMRT) sequencing approach developed by Pacific Biosciences is the first TGS approach to directly observe a single molecule of DNA polymerase as it synthesizes a strand of DNA. It does not require PCR amplification, evading amplification bias encountered in SGS. It produces longer reads lengths up to 10,000 bp, enabling easier assemblies, HLA genotyping, and even providing for the possibility of phasing entire chromosomes. The ability to capture kinetic changes, resulting from incorporation of chemical modified bases (epigenetic changes).
Much excitement has been generated with the introduction of commercial sequencer MinION in 2014. This is the only portable real-time device for DNA and RNA sequencing. This technology works by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore. Higher versions of Minion, GridIONX5, and PromethION are also available.
With newly emerging rapid and novel technologies, future of genomics seems quite exhilarating. Sequencing whole genome in less time and at reasonable cost is no longer a dream. One could expect these technologies to help unravel mysteries hidden in genome
