Table 1.

Different generations of NGS platforms.

Sr No.	Platform	Use	Sequencing Technology	Amplification Type	Principle	Read Length (bp)	Limitations	Ref.
1	454 pyrosequencing	Short read sequencing	Seq by synthesis	Emulsion PCR	Detection of pyrophosphate released during nucleotide incorporation.	400–1000	May contain deletion and insertion sequencing errors due to inefficient determination of homopolymer length.	[18–20]
2	Ion Torrent	Short read sequencing	Seq by synthesis	Emulsion PCR	Ion semiconductor sequencing principle detecting H+ ion generated during nucleotide incorporation.	200–400	When homopolymer sequences are sequenced, it may lead to loss in signal strength.	[19–21]
3	Illumina	Short read sequencing	Seq by synthesis	Bridge PCR	Solid-phase sequencing on immobilized surface leveraging clonal array formation using proprietary reversible terminator technology for rapid and accurate large-scale sequencing using single labeled dNTPs, which is added to the nucleic acid chain.	36–300	In case of sample overloading, the sequencing may result in overcrowding or overlapping signals, thus spiking the error rate up to 1%.	[19,20,22]
4	SOLiD	Short read sequencing	Seq by ligation	Emulsion PCR	An enzymatic method of sequencing using DNA ligase. 8-Mer probes with a hydroxyl group at 3′ end and a fluorescent tag (unique to each base A, T, G, C) at 5′ end are used in ligation reaction.	75	This platform displays substitution errors and may also under-represent GC-rich regions. Their short reads also limit their wider applications.	[20,23]
5	DNA nanoball sequencing	Short read sequencing	Seq by ligation	Amplification by Nanoball PCR	Splint oligo hybridization with post-PCR amplicon from libraries helps in the formation of circles. This circular ssDNA acts as the DNA template to generate a long string of DNA that self-assembles into a tight DNA nanoball. These are added to the aminosilane (positively charged)-coated flow cell to allow patterned binding of the DNA nanoballs. The fluorescently tagged bases are incorporated into the DNA strand, and the release of the fluorescent tag is captured using imaging techniques.	50–150	Multiple PCR cycles are needed with a more exhaustive workflow. This, combined with the output of short-read sequencing, can be a possible limitation.	[24,25]
6	Helicos single-molecule sequencing	Short-read sequencing	Seq by synthesis	Without Amplification	Poly-A-tailed short 100–200 bp fragmented genomic DNA is sequenced on poly-T oligo-coated flow cells using fluorescently labeled 4 dNTPS. The signal released upon adding each nucleotide is captured.	35	Highly sensitive instrumentation required. As the sequence length increases, the percentage of strands that can be utilized decreases.	[26,27]
7	PacBio Onso system	Short-read sequencing	Seq by binding	Optional PCR	Sequencing by binding (SBB) chemistry uses native nucleotides and scarless incorporation under optimized conditions for binding and extension (https://www.pacb.com/technology/sequencing-by-binding/, accessed on 1 July 2023).	100–200	The higher cost compared to other sequencing platforms.
8	PacBio Single-molecule real-time sequencing (SMRT) technology	Long-read sequencing	Seq by synthesis	Without PCR	The SMRT sequencing employs SMRT Cell, housing numerous small wells known as zero-mode waveguides (ZMWs). Individual DNA molecules are immobilized within these wells, emitting light as the polymerase incorporates each nucleotide, allowing real-time measurement of nucleotide incorporation	average 10,000–25,000	The higher cost compared to other sequencing platforms.	[28,29]
9	Nanopore DNA sequencing	Long-read sequencing	Sequence detection through electrical impedance	Without PCR	The method relies on the linearization of DNA or RNA molecules and their capability to move through a biological pore called “nanopores”, which are eight nanometers wide. Electrophoretic mobility allows the passage of linear nucleic acid strand, which in turn is capable of generating a current signal.	average 10,000–30,000	The error rate can spike up to 15%, especially with low-complexity sequences. Compared to short-read sequencers, it has a lower read accuracy.	[5,19,30]