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Benefits of NGS over first and third generation sequencing.

With the discovery of the DNA double-helical structure consisting of 4 deoxyribonucleic acids by Watson JD et al. in 1953, a stimulation to decode the puzzle of the genomic sequence of all the organisms arose. The curiosity resulted in the sequencing of 77nt yeast alanine tRNA with a proposed cloverleaf structure but unable to identify anticodon that will bind to mRNA sequence after 12 years in 1965, and another seven years to sequence 1 g of the tRNA from commercial baker’s yeast by counter current distribution. A revolutionary turn for sequencing was in 1977 when firstly, Maxam-gilbert gave the concept of chemical sequencing (published in February) and secondly, Frederick Sanger put forward chain terminator sequencing, dideoxy sequencing method (published in December). Initially, chemical sequencing gain popularity, yet dideoxy sequencing proved to be more automated, simple and reliable; hence prevailed for decades. Interestingly, even though NGS utilize the same concept of Sanger sequencing, the second generation NGS techniques can parallelly sequence millions of fragments in a single run differentiating from Sanger, which can sequence a single DNA fragment at a time.

Advancement of second-generation over the first generation of sequencing

With the labelling of di-deoxyribonucleic acid (ddNTPs) with florescent, L. Hood and M. Hunkapiller, in association with Applied Biosystems, Inc. (ABI), built a single reaction chain Automated first-generation sequencer the prism 310 gene analyser based on the concept of Sanger sequencing. Besides, the addition of computers and the invention of Taq polymerase & PCR (between 1985-1990) and Reverse transcriptase lead to the enhancement of this technology by enabling it to collect, store & analyse data, generate random and specific sequences for de novo sequencing, resequencing of region of interest and notably sequencing of RNA by converting it into cDNA Etc. Despite the improvements in 1st generation sequencing and collaboration of many laboratories worldwide, it took 15 years to complete the Whole Human genome sequencing project and coasted approximately one hundred million US dollars. In contrast, 454 Genome Sequencer FLX, a Next-Generation Sequencer (NGS), could complete in two months in one-hundredth of the cost.

The remarkable development was that researchers used the luminescent method for measuring pyrophosphate synthesis instead of inferring nucleotide identity through radio- or fluorescently-labelled dNTPs or oligonucleotides. Furthermore, in 2005, the first high throughput sequencing (HTS) machine, the 454 GS 20 machine, was developed, breaking the limitation of first-generation sequencing by the generation of millions of short reads in parallel, detection of sequence output directly without electrophoresis, thus lowering the cost and speeding up the sequencing process. Soon after the success of 454, various parallel sequencing techniques sprung up. Some of them are Illumina (Solexa) sequencing, Ion torrent (proton/PGM sequencing), Solid sequencing.

Comparison of Sanger Sequencing, NGS and Third generation sequencing

Pic Credit: Jennifer Ronholm

NGS and Third generation sequencing are complementary not successive!!!

The boom of third-generation sequencing was in 2015, and the most significant advancement over any other sequencing method is the elimination of laborious, time-consuming library preparation and direct RNA sequencing without cDNA preparation. Few more advantages include the capability to produce long reads up to hundreds of kilobase pairs, on-field real-time sequencing, and less laborious. However, the drawback of low accuracy, high error rate (14%), and scalability challenges outweigh the advantages.

Despite the enhancement in third-generation sequencing over the period, it still does not potentially replace NGS and emerge as a better candidate for sequencing related problems. Thus, Both NGS and Third-generation sequencing complement each other since the limitation of short read to successful assembly of the highly repetitive region and closed bacteria resolved by third-generation sequencing by producing long reads and sequencing several repeat regions or low GC content. Therefore, the wise strategy would be to generate a closed-genome scaffold using third-generation technology and then generate in-depth coverage error-free SNP analysis with second-generation techniques.


Heather, J. M., & Chain, B. (2016). The sequence of sequencers: The history of sequencing DNA. Genomics, 107(1), 1-8.

Key differences between next-generation sequencing and Sanger sequencing |

Kulski, J. K. (2016). Next-generation sequencing—an overview of the history, tools, and “Omic” applications. Next generation sequencing–advances, applications and challenges, 3, 60.

Ronholm, J., Nasheri, N., Petronella, N., & Pagotto, F. (2016). Navigating microbiological food safety in the era of whole-genome sequencing. Clinical microbiology reviews, 29(4), 837-857.

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