The Genome Sequencer FLX Instrument, powered by GS FLX Titanium and Standard series reagents, features a groundbreaking combination of long reads, exceptional accuracy, and high throughput. The instrument uses a system based on 454's sequencing by-synthesis technology.

How it Works

The complete sequencing workflow of the Genome Sequencer FLX System comprises four main steps:

1. Generation of a single-stranded template DNA library

2. Emulsion-based clonal amplification of the library

3. Data generation via sequencing-by-synthesis

4. Data analysis using different bioinformatics tools

Sample Input and Fragmentation

The Genome Sequencer FLX System supports the sequencing of samples from a wide variety of starting materials including genomic DNA, PCR products, BACs, and cDNA. Samples such as genomic DNA and BACs are fractionated into small, 300 to 800 basepair fragments. For smaller samples, such as small non-coding RNA or PCR amplicons, fragmentation is not required. Instead, short PCR products amplified using Genome Sequencer fusion primers can be used for immobilization onto DNA capture beads.

Library Preparation

Using a series of standard molecular biology techniques, short adaptors (A and B) - specific for both the 3' and 5' ends - are added to each fragment. The adaptors are used for purification, amplification, and sequencing steps. Single-stranded fragments with A and B adaptors compose the sample library used for subsequent workflow steps.

The single-stranded DNA library is immobilized onto specifically designed DNA Capture Beads. 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 microreactors containing just one bead with one unique sample-library fragment.

emPCR (Emulsion PCR) Amplification

Each unique sample library fragment is amplified within its own microreactor, excluding competing or contaminating sequences. Amplification of the entire fragment collection is done in parallel; for each fragment, this results 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.

Applications

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, resequencing), including shotgun sequencing (bacteria, viruses) and BAC-based shotgun sequencing (animals, plants), amplicon sequencing, and small RNA sequencing, including EST and transcriptome analysis.

Sequencing Options

There are several sequencing options available based on your project needs. For a complete list, please contact us.

Standard GS FLX Chemistry:

100Mb Shotgun Prep, Sequence $11,300

50Mb Amplicon Prep, Single Sequence $9,900

50Mb Amplicon Prep, Shared Sequence $7,300

100Mb Amplicon Prep, Sequence $10,400

Titanium GS FLX Chemistry:

2 Region Titanium Prep, Single Sequence $13,300

4 Region Titanium Prep, Sequence $15,300

Sample Requirements

The requirements are the same for both Standard and Titanium FLX chemistries

1. DNA must be double stranded.
2. DNA should not be the result of whole genome amplification (or other similar process which may compromise representativity)
3. DNA should not be degraded, i.e. starting DNA material should be in pieces >1.5kb (70-800bp for LMW DNA)
4. DNA should contain no particulate matter
5. DNA should have an OD 260/280 ratio of approximately 1.8
6. DNA sample should have a minimal concentration of 50ng/ul, in TE
7. Total DNA required: 3-10 ug.

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.

Expected Results

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

Titanium GS FLX Chemistry:

Throughput for 2-Region Plate: 360-560 million high-quality, filter-passed bases per run

Throughput for 4-Region Plate: 240-440 million high-quality, filter-passed bases per run
Read Length: Modal length = 500 bases, Average length = 400 bases Accuracy Q20 read length of 400 bases (99% at 400 bases and higher for prior bases)

Reads per run: >1 million high-quality reads

Contact Information

Please contact us for more information on this service.

Ryan Sprissler
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621-4882

Heather Issar
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621-9184