- Course overview
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- Introduction
- Real-time PCR
- Microarrays
- RNA sequencing
- Biological interpretation of gene expression data
- Genotyping, epigenetic and DNA/RNA-protein interaction methods
- DNA/RNA-protein interactions
- Summary
- Quiz: Check your learning
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- References
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454 sequencing
Roche 454 sequencing can sequence much longer reads than Illumina. Like Illumina, it does this by sequencing multiple reads at once by reading optical signals as bases are added.
As in Illumina, the DNA or RNA is fragmented into shorter reads, in this case up to 1kb. Generic adaptors are added to the ends and these are annealed to beads, one DNA fragment per bead. The fragments are then amplified by PCR using adaptor-specifc primers.
Each bead is then placed in a single well of a slide. So each well will contain a single bead, covered in many PCR copies of a single sequence. The wells also contain DNA polymerase and sequencing buffers. See Figure 8 for a representation of 454 sequencing workflow.
