Dr Jitendra K. Thakur

National Institute of Plant Genome Research

DNA Sequencing in the 21st Century

It is very important to know the genome of an organism. Scientists all over the world, including India, are investing so much in different sequencing projects. In the Earth Biogenome Project, scientists across the globe are trying to sequence genomes of all the known species. Such an ambitious initiative could be thought of simply because of the availability of powerful technology of NGS (next-generation sequencing), also known as massive parallel sequencing. After initial hiccups of automation of first-generation Sanger sequencing, the advent of NGS, followed by continuous modifications, has contributed significantly in generation of massive sequence data in shorter time and reduced cost. Arguably, NGS is considered to be the most vibrant and dynamic technology, which is growing with remarkably fast pace. The fascinating transitional journey from the first human genome sequence to the personal genome sequencing within a short span of one-and-a-half decade has been made possible only because of the advances made in NGS. However, complete annotation of the complicated eukaryotic genome sequences (repeat sequences, microsatellites, and other complicated genomic structures) still remains a big challenge.

Although a number of NGS or second-generation sequencing platforms are available, the Illumina platforms are most popular covering almost 75 percent of the world market. Illumina keeps launching new sequencing platforms with ability to produce more and more data. Currently, the company offers Mi-Seq, Next-Seq 500, Hi-Seq 2500 platforms with ability to produce 15 Gb, 120 Gb, and 1000 Gb of sequencing data per run covering maximum of 2×300 bp, 2×150 bp and 2×125 bp read lengths, respectively. HiSeq X10 has become the undisputed flagship Illumina platform, churning out 1.6–1.8 Tb sequence data per run. Generally, Illumina platforms use bridge amplification and sequencing by synthesis (SBS) technology, which is a widely adopted NGS technology responsible for generating more than 90 percent of the sequencing data worldwide. Since genomes of human and other higher eukaryotes harbor several repetitive regions, these small reads of 100–300 base pairs pose huge challenge for proper alignment and subsequent annotations, especially in de novo sequencing. So, in an effort to overcome such inherent challenges of genomics, the so-called third-generation pacific bioscience (PacBio), with long-read sequencing technology, has been introduced. This PacBio utilizes single-molecule real-time (SMRT) sequencing technology harnessing the natural process of DNA replication that enables real-time observation of DNA synthesis. SMRT sequencing is built upon two key innovations: zero-mode waveguides (ZMWs) and phosphor-linked nucleotides. SMRT sequencing is suitable for a variety of research applications, and offers many benefits including longest average read lengths, highest consensus accuracy, and uniform coverage. In the other latest advancements made in the field, MinION, the world’s first nanopore DNA sequencer is worth mentioning. It was developed by Oxford Nanopore Technologies in 2015. The MinION is a portable platform that can sequence relatively longer reads.

Thus, in today’s scenario, both Illumina HiSeq and PacBio are most preferable platforms for DNA sequencing, but they have their individual pros and cons. To minimize the tradeoff between quality and quantity, Illumina HiSeq platform is mostly preferred as the error rate in large reads generated by PacBio systems is more. In addition, PacBio platform is very expensive in comparison to Illumina platform. Even the running cost is more. In the market, the HiSeq X10 has captured most of commercial sector whereas academic research laboratories prefer HiSeq 2500/2000. However, for generating gold standard sequences for all types of commercial and academic purposes, we require both Illumina Hi-Seq 2500 (for generation of short reads up to 150 bp) and PacBio Sequel II (for the long reads up to 10 kb). This will give us option to align small reads on the long reads. Scientists and researchers all over the world are eagerly waiting for the newly emerging technology, direct sequencing of RNA without first converting it to DNA. This particular advancement will dramatically improve our ability of gene annotation in the future.

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