454 GS FLX Sequencing

The inqaba biotec GS-FLX platform is Africa’s first high-speed, complete solution, and efficient high-throughput sequencing service. This BioPad funded initiative is aimed at assisting all scientists, both locally and internationally.

The Genome Sequencer system was developed using novel 454 technology. It allows life scientists to enjoy the benefits of fast, accurate, and cost-effective high-throughput sequencing. This versatile technology enables the sequencing of many sample types, including short DNA fragments, amplicons, shotgun fragments and paired ends.

Dr Arshad Ismail, the GS Technology Platform Manager, and the inqaba biotec GS Facility

inqaba biotec is a professional and experienced team and has generated over ~7.3 GB of data since March 2007. During this time, the GS technology has been improved from an initial 20 MB per run (~100 base read length) to the 100 MB per run of the GS FLX (~250 base read length) and now the 500 MB per run of the GS Titanium (~500 base read length).

Evolution of the 454

Evolution of the 454 sequencing increasing reads/run, read length and throughput

Benefits of the GS FLX Technology:

1. Flexibility:
A broad range of applications is covered by a single versatile sequencing platform, including:

De novo Sequencing - The initial sequencing that results in the primary genetic sequence of organisms.

Resequencing – The task of sequencing a DNA region for an individual given that a reference sequence for this region is already available for the specific species.

Transcriptome Analysis - Also referred to as Expression Profiling, examines the expression level of mRNAs in a given cell population, often using high-throughput techniques such as High Throughput sequencing or DNA microarray.

Gene Regulation Studies – The study of the processes that cells and viruses use to turn the information on genes into gene products.

Epigenetic Changes – The study of changes in gene expression not as a result of nucleotide changes

Metagenomics & Microbial Diversity – The study of genetic material taken directly from environmental samples.

Paleogenomics – The application of genetics to paleontology, including ancient and degraded DNA samples.

2. Speed:

Generate more than 500 million bases in a 10 hour instrument run. Average yield is greater than 1 000 000 reads per run. The genome sequence of a 5 MB bacterial genome can be deciphered in 5 days, whereas the Sanger method usually takes months.

500 MB in 10 hours of Sequencing reaction

3. Accuracy:

GS technology generates ±1 000 000 reads of ~500 bases per reaction, and has a consensus accuracy of >99.99% due to 10-20 fold oversampling.

4. Productivity:

Use the complete system solution; from sample preparation to data mapping or assembly. Eliminate cloning, colony picking, AT bias, or problems with GC rich regions.

5. Cost Efficiency

Benefit from reduced cost per base compared to conventional Sanger Technology due to massive parallelization. Flexible sample-loading options, support various throughput and experimental design needs, enabling efficient use of reagents and consumables.

6. Easy to use

Perform sequencing runs with an easy to use instrument requiring minimal steps. Our experienced team will help you all the way through the steps of project planning to the data analysis.

Applications of GS FLX Technology:

Small RNA	List of Publications
Whole Genome Sequencing	List of Publications
BAC’s/Plastids/Mitochondria	List of Publications
Chromosome Structure/Epigenetics	List of Publications
Transcriptome Analysis	List of Publications
PCR Amplicons	List of Publications
Metagenomics and Microbial Diversity	List of Publications
Ancient DNA	List of Publications
Technology and Informatics	List of Publications
GS FLX Related Publications	List of Publications

How does GS FLX/Titanium Technology work?

Sample Input and DNA Fragmentation

Our GS FLX/Titanium sequencing platform supports sequencing from a wide variety of sample types. These include: genomic DNA, PCR products, BACs and cDNA.

5 - 10 µg of gDNA samples are nebulized into small 500 to 800 base pair fragments, using high pressure Nitrogen gas. The smaller samples, such as small non-coding RNA (siRNA) and PCR amplicons, do not require this step.

Fragmentation of the gDNA using Nitrogen Nebulisation

Library Preparation

Short specific adaptors (A and B) are ligated onto each small fragment. These are vital for the steps that follow, including purification as the B adaptor as an attached biotin label. The dsDNA fragments are purified using Streptavidin beads.

dsDNA with ligated adaptors

dsDNA purification with streptavidin bead

Purification of the dsDNA with ligated adaptors using Streptavidin beads

The single stranded fragments (sstDNA) with the adaptors that are released from the streptavidin beads are quantified for the emPCR™ reaction by titration.

emPCR™

The sstDNA library is added to tubes with capture beads and thermostable water-in-oil emulsion, with approximatley 2.4 million beads. The water-in-oil mix is shaken up using a tissue lyser to create an emulsion resulting in each bead being captured into its own microreactor. Each Microreactor contains a bead, a single sstDNA fragment and the complete PCR amplification mix.

The water-in-oil emulsion showing the microreactors

The emulsion is aliquoted into PCR plates for amplification. Clonal amplification of microreactors occurs simultaneously, and each bead ends up with ~10⁶clonal copiesof the sstDNA.

A clonally amplified bead with ~10⁶copies of the sstDNA

The Sequencing reaction

The GS FLX Titanium PicoTitrePlate™ has been modified and now has a metal coating that prevents cross talk between the wells. There are approximately 3.4 million wells per plate and each have a diameter of 29 µM. This ensures that only 1 bead (diameter of 20 µM) is able to fit into each well.

The GS Titanium PicoTitrePlate™

The clonally amplified sstDNA beads are added to the DNA Bead incubation Mix which contains DNA polymerase. This is layered with Enzyme beads (containing sulfurylase and luciferase) onto the PicoTitrePlate™ device. The loaded device is centrifuged and the beads are deposited into the wells.

Loading of the PicoTitrePlate™

Once loaded, the PicoTitrePlate device is placed in the GS instrument against a powerful CCD array.

Loading the PicoTitrePlate™ into the GS instrument

Sequencing reagents (containing buffers and nucleotides) are flowed across the wells of the plate by the fluidics sub-system. Separate nucleotides are flowed across the plate in a fixed order, allowing each of the thousands of beads with millions of copies of DNA to be sequenced in parallel. As a nucleotide complementary to the template strand is added, the DNA polymerase incorporates it into the growing DNA strand. This results in a light reaction and is recorded by the CCD camera in the instrument. The intensity of the light signal is directly proportional to the number of nucleotides (i.e. homopolymer stretches) that are incorporated in a single flow.

Sequencing by synthesis using the GS Titanium

The sequencing by synthesis reaction

Data and analysis:

Data is stored on site at inqaba biotec and is sent to you in several formats depending on customer requests. We perform the initial assemblies and alignments. Data analyses is taken further on customer request. We often have our customers visiting us to learn how to work with the data.

Many bioinformatics programs are available, and several are freeware, unfortunately, most of these are not able to deal with the volume of data. We recommend that our customers use the CLC bio Genomics Workbench software. This is a complete bioinformatics solution and is the only software able to cope with data coming off all of the different High throughput sequencing platforms.

Article on New Generation Sequencing and CLC Bio