Table 1 A comparison of features, chemistry and performance of first, second and third generation sequencing platforms. CMOS Complementary metal-oxide semiconductor, TH throughput, SM single molecule, SMRT single molecule real time
From: Next-generation sequencing and its potential impact on food microbial genomics
Platform | Chemistry | PCR amplification | Starting DNA | Read length | Reads/run | TH/run | Run time | Disadvantages | Applications | References |
|---|---|---|---|---|---|---|---|---|---|---|
Sanger sequencing | Asynchronous with base-specific terminator | Standard PCR | 0.5-1 mg | 700 | Few 1,000 bp | 1 Mb | 2 h | PCR biases; low degree of parallelism; high cost of sequencing | Gene/genome sequencing | |
Roche 454 | Sequencing-by-synthesis (Pyrosequencing) | EmPCR | 1 μg for shotgun library and 5 μg for pair-end | > 400 | 1,000,000 | 0.4-0.6Gb | 7-10 h | PCR biases; asynchronous synthesis; homopolymer run; base insertion and deletion errors; emPCR is cumbersome and technically challenging | De novo genome sequencing, RNA-seq, resequencing/targeted re-sequencing | http://www.454.com/; Mardis 2008a; Metzker 2010 |
Illumina | Polymerase-based sequencing-by-synthesis | Bridge amplification | <1 μg for single or pair-end | 75/2 × 100a | 40,000,000 | 3-6/200* Gb | 3-4 days | PCR biases; low multiplexing capability of samples | De novo genome sequencing, RNA-seq, resequencing/ targeted re-sequencing, metagenomics, ChIP | http://www.illumina.com/technology/sequencing_technology.ilmn; Mardis 2008a; Metzker 2010 |
SOLiD | Ligation-based sequencing | Em PCR | <2 μg for shotgun library and 5-20 μg for pair-end | 35-40 | 85,000,000 | 10-20Gb | 7 days | EmPCR is cumbersome and technically challenging PCR biases; long run time | Transcript counting, mutation detection, ChIP, RNA-seq etc. | http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing.html; Mardis 2008a; Metzker 2010 |
HeliScope | Polymerase (asynchronous extension) | SM; no PCR | <2 μg, single end only | 25-50 | 1,000,000,000 | 28Gb | 8 days | Asynchronous synthesis; homopolymer run; high instrument cost; short read lengths; high error rates compared with other reversible terminator chemistries | Resequencing, transcript counting, ChIP, RNA-seq | http://www.helicosbio.com/Products/HelicosregGeneticAnalysisSystem/HeliScopetradeSequencer/tabid/87/Default.aspx Metzker 2010 |
Polonator | Synchronous controlled synthesis | Em PCR | __ | 26 | 160,000,000 | 4.5Gb | 4 days | Low read length; emPCR is cumbersome and technically challenging | Bacterial genome, resequencing, SNPs and structural variants detection | Metzker 2010 |
PacBio | Phospho-linked fluorescent Nucleotides | SMRT | ∼1.5 μg (ideally 2-3 μg) | 1,000-1,200 | 100,000,000 | 100Gb/Hr | 8 h | High instrument cost; low number of sequence read per run; highest error rates compared with other NGS chemistries | De novo genome sequencing, RNA-seq, resequencing/targeted re-sequencing, metagenomics, SNPs and structural variants detection | |
http://www.pacificbiosciences.com/products/smrt-technology/smrt-sequencing-advantage | ||||||||||
http://www.pacificbiosciences.com/partner_products/PartekGenomicsSuite_2.7.12.pdf; Travers et al. 2010 | ||||||||||
Metzker 2010 | ||||||||||
CMOS non-optical sequencing | Template-directed DNA polymerase synthesis | __b | __ | __ | __ | __ | __ | __ | De novo genome sequencing | Rothberg et al. 2011 |