Next Generation Sequencing
Rx Biosciences offers 454 sequencing using the Genome Sequencer FLX Instrument, powered by GS FLX Titanium and Standard series reagents. The high throughput sequencing technology features a ground breaking combination of quick, long readouts with exceptional precision in a high throughput manner.
Steps involved in 454 sequencing
- Preparation of a single-stranded template DNA library
- Emulsion-based amplification of individual clones of the library
- High throughput sequence data generation via sequencing-by-synthesis
- Data analysis using different bioinformatics tools, contig generation and assembly into long sequences
Sample Input and Fragmentation
Wide variety of starting materials including genomic DNA, PCR products, BAC clones, and cDNA can be used for 454 FLX sequencing. Samples such as large genomic DNA and BAC clones are fragmented into small, 300 to 800 base-pair fragments where as for smaller templates, such as small non-coding RNA or PCR amplicons, fragmentation is not needed. Instead, short PCR products amplified using Genome Sequencer fusion primers are used for immobilization onto DNA capture beads.
300-800 base-pair fragments are blunted and ligated with 5’ and 3’ specific unique adapters which are used further in down stream purification, amplification, and sequencing steps. Single-stranded fragments with 5’ and 3’ specific adapters constitute the sample library and used for subsequent immobilization and sequencing steps.
The single-stranded DNA library is immobilized onto specifically designed DNA Capture Beads in such a way that each bead carries a unique single-stranded DNA library fragment. The bead-bound library is emulsified with amplification reagents in a water-in-oil mixture resulting in micro-reactors (vesicles) containing just one bead with one unique sample-library fragment.
Emulsion PCR (emPCR) Amplification
Each unique sample library fragment is amplified within its own micro-reactor, excluding competing or contaminating sequences. Amplification of the entire fragment collection is done in parallel; for each fragment, this result in a copy number of several million per bead. Subsequently, the emulsion PCR is broken while the amplified fragments remain bound to their specific beads.
The clonally amplified fragments are enriched and loaded onto a PicoTiterPlate device for sequencing. The diameter of the PicoTiterPlate wells allows for only one bead per well. After addition of sequencing enzymes, the fluidics subsystem of the Genome Sequencer FLX Instrument flows individual nucleotides in a fixed order across the hundreds of thousands of wells containing one bead each. Addition of one (or more) nucleotide(s) complementary to the template strand results in a chemiluminescent signal recorded by the CCD camera of the Genome Sequencer FLX Instrument.
The high throughput 454/GS FLX Standard and 454/GS FLX Titanium sequencing technologies have many applications. Some of the most common are whole genome sequencing (de novo, re-sequencing), including shotgun sequencing (bacteria, viruses) and BAC-based shotgun sequencing (animals, plants), amplicon sequencing, and small RNA sequencing, including EST and transcriptome analysis.
- 2-10 ug of high molecular weight (>1.5 kb) double stranded DNA.
- DNA should have an OD 260/280 ratio of approximately 1.8 to 2.0.
- DNA sample should have a minimal concentration of 50ng/ul, in T
Quality Control of Samples
Every sample received for sequencing will go through a strict quality control check before it is processed. A PicoGreen Assay will be preformed to verify that there is the correct amount of starting DNA. After the sample has been quantified, it will be run on a Bioanalyzer DNA 7500 chip to check the quality of DNA and verify that there is minimal degradation. Customers will be asked for more DNA if their sample fails either of the check points.
Standard GS FLX Chemistry
- Throughput for 50Mb Plate: 50 million high-quality filter-passed bases per run
- Throughput for 100Mb Plate: 100 million high-quality filter-passed bases per run
- Read Length & Accuracy: 250 bases with >99.5% single-read accuracy
- Reads per run: up to 400,000 high-quality reads
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