Next-Generation Sequencing, an innovation in science

By Sadaf Tariq (M.Phil Scholar)
Ambreen Zahra (M.Sc Scholar)

Next-Generation Sequencing, an innovation in science

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Abstract

Next-generation sequencing (NGS) is also known as high throughput sequencing. The Human Genome Project was completed by using Sanger sequencing in over 10 years and its cost was nearly $3 billion (Sanger et al., 1977). NGS is an innovative sequencing method during which millions of parallel reactions take place for high throughput sequencing. Next-generation sequencing makes it easier to sequence the whole genome in a single experiment. With its ultra-high-speed, scalability, and throughput, NGS has completely revolutionized biological sciences. This parallel sequencing technique is used to study genomes at the next level.  NGS has filled the gap to address all the complex genomics questions and has become an everyday research technology. With innovative data analysis options, researchers can easily sequence the whole genome within a day. NGS is also playing its role to uncover the genetic susceptibility of mildly ill COVID-19 patients.

Introduction

DNA sequencing is a process to determine the nucleotides sequence of the targeted area in DNA. Sanger sequencing was the first commercialized method of DNA sequencing introduced in 1977 and remained dominated for many decades. It requires a single-stranded DNA template, DNA primers, DNA polymerases, dNTPs, and modified ddNTPs. These modified ddNTPs lack 2' and 3' hydroxyl group, required for the formation of a phosphodiester bond between two nucleotides. Hence, it ceases the extension of DNA. These are radioactively or fluorescently labeled for detection. NGS is at an advantage to conventional techniques for its high throughput and massively parallel sequencing at a reduced cost. Different technologies are involved in NGS: Illumina sequencing works by simultaneous detection by the addition of nucleotides and emits unique fluorescent signals upon the addition of labeled nucleotides. Roche sequencing detects the release of phosphate group as fluorescence is emitted and new nucleotide is added to the existing chain. Ion semiconductor sequencing detects the release of protons upon the incorporation of individual nucleotides by DNA polymerases. It does not emit any light signal (Yohe&Thyagarajan, 2017).

Applications of NGS

·         To sequence whole genome

NGS platforms are used to perform the whole genome sequencing. Millions of small fragments of the genome are sequenced in parallel. There is a wide variety of platforms using different sequencing technologies. Bioinformatics tools are used to analyze the sequenced data and join the individual reads to get a final draft (Ansorge, 2009).

·         To sequence only specific regions of the genome

Exons, introns, repeated sequences, tandem repeats are sequenced, identified and detected by NGS. Open reading frames and non-open reading frames can also be sequenced and identified (Adams &Eng, 2017).

·         To sequence cancer subclones and other rare variants

Single-cell techniques can be used to study the cell microenvironment, interpret all the gene expression patterns provide insights on drug resistance patterns and metastasis (Aravanis et al., 2017).

·         To discover susceptibility of novel pathogens

Each of the three billion bases are sequenced multiple times for high depth insight into DNA variation and provides accurate data on unexpected variations in the genome. A full spectrum of genomic variation can be obtained by this technology (Ansorge, 2009).

·         To sequence and map whole-exome

NGS is also used to sequence constrained areas of genome for better interpretation of gene position and locality. NGS is able to sequence and map all 22,000 coding genes (whole-exome sequencing) or only a specific numbers of individual exomes.

·         To sequence transcriptome for uncovering rare diseases

NGS can help us to study the transcriptome of high-risk populations. It can uncover rare diseases, genetic susceptibility, and their risk factors. Thus, preventive measures can be taken earlier to prevent diseases (Adams &Eng, 2017).

·         To assess risk factors of COVID-19

NGS is being used by UK researchers to compare sequenced genomes of COVID-19 patients. It is uncovering the genetic susceptibility of patients by revealing all the involved genetic factors (Lu et al., 2020).

Methodology

Following common steps are followed during all NGS techniques:

1.      Sample Preparation

DNA is used as a starting material. Only pure DNA can lead to precise and accurate results. Spin column DNA extraction is the most preferable extraction technique of all. About 100ng DNA is used for the reaction. The extracted DNA is further amplified by PCR assay. The amplicons are run on 2% agarose gel with DNA ladder. Spin column PCR purification is done after agarose gel electrophoresis for amplicons purification as the amplicons may be contaminated with unbound primers, dimers, unused templates, and unused buffers. These contaminants can abort sequencing. The sample preparation for NGS requires a genome library which can be obtained either by ligation or amplification to an adapter sequence. These adapter sequences are crucial to provide a universal priming site for sequencer primers. These adapters also work as sequencing chips during hybridization (Ronaghi, 2001; Shendure&Ji, 2008; McCombie et al., 2019).

2.      Library preparation

The genomic library is prepared by fragmentizing the whole genome by restriction nucleases. The whole-genome DNA fragments are joined with known DNA fragments. The ligation is followed by the addition of adapters at the terminals and the process is adapter ligation. Only the ligated fragments are added in the genomic library. The unbound sequences are washed away by washing buffers (Mardis, 2008; Slatko et al., 2018).

3.      Cluster generation

The fragmented DNA is loaded on the immobilized oligos on the surface. The clusters of DNA are generated by bridge amplification. The DNA fragments are bent over one adapter sequence by one end and bind to another sequence by another end. The primers bind to the DNA and amplification is occurred vertically. The bridge amplification results in the generation of two new DNA strands.

4.      Sequencing

The polymerases add the nucleotides into the bridge amplification; the sequencing is the next step. The reaction tubes containing amplicons are placed into a sequencer machine. A number of cycles of denaturation, annealing, and extension take place simultaneously in the sequencer. The labeled nucleotides produce signals during the addition reactions which are recorded by a computer (Shendure & Ji, 2008). Each library fragment undergoes amplification on a solid matter surface. These surfaces can be either bead or silicon derived sheets with DNA linkers used to hybridize library adapters. After repeated cycles of nucleotide incorporation, amplification results in formation of clusters of DNA, each of them acts as an individual reaction. These clusters are sequenced and read optically in the form of light or fluorescent signals. All these signals are sent to the computer for data analysis.

5.      Data output

Each cycle of nucleotide incorporation provides data about the complete sequencing draft. This data is collected and further analyzed for more meaningful results. The bioinformatics based inbuilt software process the file saved after DNA sequencing and compare it with available data for analysis. After data analysis, the software can interpret the variations and mutations in any gene (Slatko et al., 2018).

Table: Basic features of NGS platforms

NGS Platforms	Read length per run (bp)	Time	Error rate(%)	Chemistry
Illumina sequencing	2x300	27 h	0.8	Reversible terminators
Roche sequencing	700	24 h	1	Pyrosequencing
Ion semiconductor sequencing	200	2-5 h	1	Proton detection sequencing
SOLiD sequencing	50	14 days	0.01	Sequencing by ligation
Nanopore sequencing	>5000	48 h	1	Real-time sequencing

 

 

Future Prospects

NGS has revolutionized biological sciences, medical sciences, and health sciences. It is being used to diagnose rare diseases, multiple genetic disorders, and syndromes. Optimizations and advancements are reducing the error rate and improving the cost and time issues. Semiconductor sequencing is revolutionizing with CMOS technology. It is eager to provide high accuracy data by one channel SBS. The two-channel SBS is even faster sequencing than original SBS version with high throughput and even higher accuracy. The NextSeq 2000 the system is working for new emerging applications to analyze all the data in less than 2 hours (Brink et al., 2019).

 Author's Details

Sadaf Tariq 1, Ambreen Zahra 2

1M.Phil Scholar (Biochemistry), 2 M.Sc Scholar (Biochemistry)
1Department of Biochemistry, GC University, Faisalabad, Pakistan.

2Institute of Biochemistry and  Biotechnology, University of Punjab, Lahore, Pakistan. 

Reviewed & Edited by

Aysha Yasmin1*, M.Ahsan ul Haq 2*

1 Ph.D. Scholar,2 M.Phil Scholar

*Department of Biochemistry, GC University, Faisalabad, Pakistan.

References

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