Roche (454) General Information

Welcome to the Roche (454) Genome Sequencer FLX+ System page. On this page you will find information on system capabilities, links to useful resources, detailed pictures and explanations of important components of the system.

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James Speers

Josh Hyman, Director

1250 Biotechnology Center

425 Henry Mall Madison WI 53706

Phone: 608-262-4657 FAX: 608-265-6573

E-Mail: nextgen-seq@biotech.wisc.edu

General Information, System Capabilities and Links

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OVERVIEW
The GS FLX+ works on the basic principle that One Fragment = One Bead = One Read. What this means is that a single fragment from a DNA library will be bound on a single DNA Capture Bead then clonally amplified using emulsion PCR. That single bead will end up in a well of a PicoTiterPlate device and be sequenced, resulting in a single read from that bead. In one reaction this is done millions of times over resulting in millions of reads.

Sequencing on the GS FLX+ is based on a sequencing-by-synthesis technology:
- Nucleotides are flowed sequentially in a fixed order across the PicoTiterPlate device during a sequencing run.
- During the nucleotide flow, hundreds of thousands of beads each carrying millions of copies of a unique single-stranded DNA molecule are sequenced in parallel.
- If a nucleotide complementary to the template strand is flowed into a well, the polymerase extends the existing DNA strand by adding nucleotide(s).
- Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument.
- The signal strength is proportional to the number of nucleotides incorporated in a single nucleotide flow.
The GS FLX+ combines long reads, accuracy and high throughput into one sequencing run. The system is capable of generating more than 1,000,000 passed-filter reads with a mode read length of 700 high-quality bases. A full sequencing run can be completed in 23 hours.
SYSTEM CAPABILITIES
The GS FLX+ is suitable for a vast number of sequencing projects, a majority of which can be seen below.
- Whole genome/shotgun (prokaryotic, eukaryotic and de novo genome assemblies)
- Transcriptome
- Small RNA
- ChIP-seq, methylation analysis (bisulphite sequencing) and epigenetic studies
- Metagenome
- Mitochondria, plastids, plasmids and viruses
- Sequence capture (Nimblegen Arrays) and targeted region/enrichment
LINKS

454/Roche GS FLX+ Product Website

454/Roche GS FLX+ Peer-Reviewed Publications

How it works: Concise presentation from DNA to assembly

Pictures and Descriptions

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2, 4, 8 and 16 Region Gaskets

Gaskets are used to divide a PicoTiterPlate (PTP) into usable regions. Either the 2, 4, 8 or 16 region gasket must be used in a sequencing run.
PicoTiterPlate (PTP)

The PicoTiterPlate (PTP) contains approximately 1.7 million hexagonal wells that measure 44µm at their widest point. The width of the wells is about 1.5 times that of DNA Capture Beads, thus allowing only a single bead to deposit per well with the remaining spaces being filled by smaller Packing Beads and Enzyme Beads. The picture above is a previously-sequenced PTP that had been loaded using a 16-region gasket and BDD.
Bead Deposition Devices (BDDs)

Loading beads into a PicoTiterPlate (PTP) requires a Bead Deposition Device (BDD). There are four BDDs specifically designed to load beads into PTPs that are divided by 2, 4, 8 or 16 region gaskets.
Loading a PTP into a BDD

Either a 2, 4, 8 or 16 region Bead Deposition Device (BDD) is loaded with a PicoTiterPlate (PTP) and then the required gasket (2, 4, 8 or 16 region) is placed over the top of the PTP (see top picture). The top of the BDD is placed over the gasket and the handles on the BDD are closed, pressing the gasket firmly against the PTP and providing a liquid-tight seal between regions (see bottom picture). The top to the BDD has port holes that allow deposition of the various bead layers onto the PTP. The top picture is of a 16 region BDD without a BDD and the bottom picture is the same 16 region BDD with the BDD top.
Roche GS FLX Instrument

The Roche GS FLX instrument.
GS FLX Instrument Fluidics Area

The fluidics area, located on the right side of the GS FLX instrument, holds the reagents used during sequencing. The reagents are drawn up through sipper tubes (the blue straws in both pictures), conveyed to the left side of the instrument and flowed across the loaded PTP.
GS FLX Instrument Camera and PTP Loading

The left side of the GS FLX instrument houses the camera. Opening the camera door reveals the camera face (seen at the top of all the pictures above) and the area where the PTP is placed for sequencing. The PTP is slid into place with the opening of the wells facing down on top of peltier blocks. A gasket (seen best in the top picture above; thin and white) is placed around the petlier blocks and is sandwiched against the PTP once it is tightly secured (as in the bottom picture above). The gasket provides a tight seal, allowing reagents to be flowed across the PTP via ports at the tops and bottoms of the peltier blocks.

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General Information, System Capabilities and Links

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