Table 8.
Comparison of commercially available sequencing platforms.
| (a) Massive parallel clonal amplification with optical detection | |||
|---|---|---|---|
| Roche 454 | Life Technologies SOLiD | Illumina | |
|
| |||
| Library amplification | emPCR* | emPCR* | On glass |
| Sequencing | Incorporation of unlabeled dNTPs | Ligase-mediated addition of fluorescent oligoNTPs (2bp) | Incorporation of end-blocked fluorescent dNTPs |
| Detection | Light emission from release of PPi | Fluorescence emission from ligated dye-labeled oligoNTPs | Fluorescence emission from incorporated labeled oligoNTPs |
| Progression | Unlabeled dNTPS added in base-specific fashion | Chemical cleavage removes dye and oligoNTP | Chemical cleavage fluorescent dye and blocking group |
| Errors | Insertion/deletion | End of read | End of read |
| Length | 400 bp | 75 bp | 150 bp |
| Overall yield/run | 500 Mbp | >100 Gbp | 200 Gbp |
|
| |||
| (b) Fluorescent and semiconductor single molecule sequencing | |||
|
| |||
| Helicos | Pacific biosystem | Iontorrent | |
|
| |||
| Library amplification | N/A-tSMS** | N/A-SMRT*** sequencing | Optional PCR |
| Sequencing | Incorporation of fluorescent labeled dNTPs | Polymerase incorporation terminal phosphate labeled dNTPs | Polymerase incorporation of dNTPs releases H+ |
| Detection | Laser-induced emission from incorporated dNTP | Real time detection of fluorescent dye in polymerase active site | Semiconductor ion sensor detects H+ released during dNTPs incorporation |
| Progression | Chemical cleavage of dNTP fluorescent group | N/A fluorescent dyes are removed as PPi with dNTPs incorporation | H+ signal during each dNTP incorporation is converted in voltage signal |
| Errors | Insertion/deletion | Insertion/deletion | Insertion/deletion |
| Length | 35 bp | 1000 bp | 200–400 bp |
| Overall yield/run | 21–37 Gbp | >100 Gbp | 1 Gbp |
*emPCR (emulsion PCR) is an amplification method where DNA library fragments are mixed with beads and PCR reagents in an oil emulsion that allows massive amplification of bead-DNA in a single reaction.
**tSMS: true Single Molecule Sequencing.
***SMRT (Single Molecule Real Time)
bp: base pair, Mbp: Mega base pair (106 bp), Gbp: Giga base pair (109 bp), dNTP: deoxynucleotide-tri-phosphate, PPi: pyrophosphate.