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Next Generation Sequencing

Next generation sequencing (NGS) or second generation sequencing is the massively parallel sequencing in any of the high-throughput approaches to DNA sequencing. These technologies started to emerge in late 1990's and are commercially available since 2005. New NGS platforms have capability to generate 1 million to 43 billion short reads in a single run. A number of different NGS platform are available commercially with a unique sequencing chemistry and engineering configurations. Major NGS platforms with their basic features is shown in table below (Source Wikipedia).

 

Platform Template Preparation Chemistry Max Read length Run Times (days) Max Gb per Run
Roche 454
Clonal-emPCR
Pyrosequencing
400‡
0.42
0.40-0.60
GS FLX Titanium Clonal-emPCR Pyrosequencing 400‡ 0.42 0.035
Illumina MiSeq Clonal Bridge Amplification
Reversible Dye Terminator
2x300
0.17-2.7
15
Illumina HiSeq Clonal Bridge Amplification
Reversible Dye Terminator
2x150
0.3-11
1000
Illumina Genome Analyzer IIX Clonal Bridge Amplification
Reversible Dye Terminator
2x150
2-14
95
Life Technologies SOLiD4 Clonal-emPCR
Oligonucleotide 8-mer Chained Ligation
35-50
4-7
35-50
Life Technologies Ion Proton Clonal-emPCR
Native dNTPs, proton detection 200 0.5 100
Complete Genomics Gridded DNA-nanoballs Oligonucleotide 9-mer Unchained Ligation 7x10 11 3000
Helicos Biosciences Heliscope Single Molecule Reversible Dye Terminator 35‡ 8  25
Pacific Biosciences SMRT Single Molecule Phospholinked Fluorescent Nucleotides 10,000 (N50); 30,000+ (max) 0.08 0.5

 

 

NGS sequencing is significantly cheaper and quicker with less amount of DNA required for an accurate and reliable sequencing. The majority of processes are automated which means less labor cost and reagents required for NGS are also less as compared to first generation Sanger sequencing.

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