Overview
This lecture reviews the development and mechanics of next-generation DNA sequencing technologies, focusing on Roche 454, Illumina HiSeq 2000, and Applied Biosystems SOLiD systems.
Roche 454 Genome Sequencer FLX
- 454 simplified the Sanger sequencing preparation and launched commercially in 2005.
- Uses adapter-flanked DNA fragments attached to etched fiber chips for sequencing.
- Employs emulsion PCR amplification to replicate strands and attach them to beads.
- Beads are loaded onto a picotiter plate, filling individual wells for detection.
- Packing beads help the spectrometer read the signal from each well.
- Can process many samples in parallel, boosting throughput over Sanger.
- Has trouble accurately reading homopolymers, resulting in higher error rates.
- Sequencing cost per base is higher than some competitors.
Illumina HiSeq 2000 Sequencer
- Uses chain termination sequencing, like Sanger, with engineering improvements for highest market output.
- DNA is fragmented and size-selected (200–300 bp) using gel electrophoresis.
- Utilizes automated cluster generation on a flow cell covered in adapter sequences.
- Bridge amplification produces many copies of each fragment in clusters.
- Camera captures fluorescent signals as each base is added during sequencing.
- Compiles sequence data from images of roughly 150 million clusters per flow cell.
- Offers highly efficient, high-throughput sequencing.
Applied Biosystems SOLiD System
- Offers flexible genome sequencing for various applications.
- Steps include enzyme/sample prep, PCR/substrate prep, ligation, imaging, and data analysis.
- Only enzyme/sample prep and data analysis vary per application.
- Uses emulsion PCR and distributes fragments onto microbeads of varying sizes.
- Slides can have different numbers of sections to suit specific applications.
- Fluorescence is detected when fragments are ligated onto single-stranded sequences.
- Uses X-ActiCol chemistry with an 8-base interrogation system and 4 fluorescent primers.
- Can precisely detect single-base insertions, deletions, and SNPs.
Key Terms & Definitions
- Emulsion PCR — PCR method where amplification occurs in tiny oil droplets, isolating each DNA molecule.
- Homopolymer — A sequence of identical nucleotides, which can be difficult to sequence accurately.
- Bridge Amplification — Technique for creating clusters of identical DNA fragments on a surface.
- Flow Cell — A flat platform used in sequencing to hold DNA samples for analysis.
- SNP (Single Nucleotide Polymorphism) — A variation at a single base pair in a DNA sequence.
- Ligation — Process of joining two DNA strands together.
Action Items / Next Steps
- Review differences between Roche 454, Illumina HiSeq, and SOLiD systems.
- Study error types (e.g., homopolymer errors) and sequencing chemistry for exams.