Next Generation Sequencing

Overview
Sanger sequencing (chain termination methodology), created during the mid-1970’s by Frederick Sanger and co-workers has been the molecular biology workhorse for many years. The technique, based predominantly on separation of extended fragments of DNA with the addition of di-deoxynucleotides. These nucleotides differ from standard deoxynucleotides as they lack a 3’-OH group. Thus, chain termination. The technique has become saturated as improvements over the years such as incorporation of fluorescently tagged di-deoxynucleotides, improved read-length up to 1000 bases, incorporation of capillary electrophoresis, increased automation and higher throughput have all allowed the technique to be used in genome projects and as a common molecular tool.

However, increased bases sequenced at a reduced cost have always been desired. For this reason new theories were developed in the late 90’s. These have come to fruition in the last 5 years or so with advances in chemical and physical technology. Thus, we have now entered the next generation era of sequencing.

Methods
Three main companies occupy the bulk of the next generation sequencing market.

These are

  • 454/Pyrosequencing (Roche)
  • SOLiD (Applied Biosystems)
  • Solexa (Illumina)

454
454 sequencing was one of the first companies to take advantage of the new technology. A graphical illustration of the process is shown below. Essentially, DNA is fragmented, joined to adapters at either end of the fragmented DNA, amplified in an emulsion PCR (includes 1 μm agarose bead with complimentary adaptors to fragmented DNA), PCR amplified allowing up to 1 million identical fragments around one bead and finally dropped into a PicoTitreTube (PTT). It is here where the reaction of fluorescence occurs with the addition of nucleotides. The intensity is read proportional to the number of homo-polymeric bases added.

SOLiD
Sequencing by Oligo/Ligation and Detection is a method by Applied Biosystems. It has a similar principle to pyrosequencing as the amplification of fragmented DNA on an agarose bead is repeated. From here-on, the procedure differs. The incorporation of a ligase and universal oligonucleotides allows all possible di-nucleotides to be accounted for. Via a process of addition of a random primer, nucleotide readings at regular intervals are possible. Removal and repeating with an n-1 primer allows further bases to be identified. This procedure is repeated a number of times to deduce the sequences of the fragmented DNA.

Illumina
Illumina/Solexa methodology works in a slightly different way. However, again the principles are to amplify a fragment of DNA to allow efficient reading. The process works on breaking up DNA, adding adaptors, but this time attach not to a bead but to a slide. Fold-back PCR is then used to amplify the fragmented DNA into a cluster. Sequential addition of nucleotides are added using a polymerase.

Applications

  • Genomics
  • Transcriptomics
  • Epigenomics
  • Interactomics

The applications of next-generation sequencing is not set to typical sequencing as we know it. The shear volume of data generated now means we can sequence genomes within days and so data storage and analysis becomes a key issue. De novo sequencing is now possible at a greater level. Genome sequencing is typically not completed and gaps are left open (or often completed with Sanger sequencing). The focus is on gene discovery (organism biology) or SNP (epidemiology or evolution). The shear volume of data allows greater comparative genomics to be performed e.g. methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus (Francois et al, 2007, Future Microbiology). Re-sequencing allows specific areas to be checked, especially as a cost effective way of discovering SNPs e.g. Bacillus subtilis re-sequencing identified new mutations and suppressor mutations (Srivatsan et al, 2008, PLOS Genetics). Sequencing can be used for typing e.g. SNP and small (1-2 bp) indels in Caenorhabditis elegans (Hillier et al, 2008, Nature Methods) and HIV clinical isolates identified rare members of the viral population (Hoffmann et al, 2007, Nucleic Acids Res). Genomics can also be used for metagenomics to identify the population of bacteria and their abundance within uncultured, unpurified and/or viral population e.g. microbial census of human intestine (Gill et al, 2006, Science) and RNA viral community in human faeces (Zhang et al, 2006, PLOS Biol).

In addition to classical genomics applications, next generation sequencing has been linked to transcriptomics, epigenomics and interactomics. Methods such as SAGE (Serial Analysis of Gene Expression) have been linked to next generation sequencing – 5’ end SAGE (Hashimoto et al, 2009, PLOS One). In addition RNA-Seq has been developed where RNA is converted back to cDNA, addition of adaptors and sequenced to deliver a digital output of expressed components and their abundance – RNA-Seq (Wang et al 09, Nature Reviews). Interactomics has been applied to next generation sequencing e.g. ChIP-Seq, STAT1 (Robertson et al 2007, Nature Methods) and Histone binding (Barski et al 2007, Cell). Next generation sequencing has also been linked to epigenomics with methylation e.g. Lister & Ecker ,2009 Gen Res.

The use of next-generation sequencing has been increasing exponentially in the last few years;
There are many future challenges ahead with next generation sequencing. Increased read length, less costs and less errors are always on the radar. The analysis techniques are always being improved with new algorithms developed all the time. In addition, we are now seeing newer sequencing theories being developed, so called, next-next generation sequencing.

References
454
SOLiD
Illumina

The Sanger DNA Sequencing Methods

DNA sequencing refers to the methods and technologies that used to determine the orders of nucleotide bases in a DNA molecule, namely adenine (A), guanine (G), cytosine (C) and thymine (T). DNA sequencing enables us to perform a thorough analysis of DNA because it provides us with the most basic information of all: the sequence of nucleotides. The knowledge of DNA sequences has formed the basis of basic biological researches and clinical genetic diagnosis. There are also numerous applied technology fields such as biotechnology, forensic science and biological systematics that are heavily dependent on the information generated through DNA sequencing.

The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the human genome, in the Human Genome Project. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences the genomes of many animals, plants, and microorganisms.

The Sanger method, also referred to as dideoxynucleotide sequencing or chain termination sequencing, is based on the use of dideoxynucleotide (ddNTP) in addition to the normal nucleotides (dNTP) found in DNA. Dideoxynucleotide are essentially the same as nucleotides except they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH). These modified nucleotides, when integrated into a DNA sequence, prevent the addition of further nucleotides thus stop the elongation of the DNA chain. This occurs because a phosphodiester bond cannot form between the dideoxynucleotide and the next incoming nucleotide, and thus the DNA chain is terminated.The Sanger methods

Although the various reagents, equipment and methodological strategies for carrying out DNA sequencing have undergone constant evolution to improve the simplicity, speed and reliability of the process, the basic procedure which all modern gel-based enzymatic DNA sequencing utilizes has not changed over the decades since its invention. Following steps are the outlines of the procedures of the Sanger methods:

  1. The region of DNA to be sequenced is amplified in some way and then denatured to produce single stranded DNA.
  2. A sequencing primer is annealed to the single stranded DNA.
  3. Dideoxynucleotide chain termination DNA sequencing then takes advantage of the fact that a growing chain of nucleotides, extending in the 5’ to 3’ direction, will terminate if, instead of a conventional deoxynucleotide, a 2’3’ dideoxynucleotide becomes incorporated. By performing four separate reactions, each containing a DNA polymerase and a small amount of one of the four dideoxynucleotides in addition to all four deoxynucleotides, four separate sets of chain-terminated fragments can be produced.
  4. Following the replication/termination step, these chain terminated fragments will remain bound to the single stranded DNA molecule which has acted as a template. By heating these partially double stranded molecules and adding a denaturing agent such as formamide, the single stranded chain termination molecules can be released from their template and separated using high resolution denaturing gel electrophoresis.
  5. The sequence of the original region of DNA is then finally deduced by examining the relative positions of the dideoxynucleotide chain termination products in the four lanes of the denaturing gel.

Computer Telepathy Could Help Patient and Gamers

It seems the age of telepathy is here – or at least it is when you hook up two people to their computers through the internet. Scientists at the University of Southampton in England have successfully beamed a message between two people, who didn’t have to say a word to communicate it.

How it works is quite simple. One person is strapped to an Electroencephalography (EEG) amplifier by electrodes stuck to their scalp – this measures the electrical activity in their brain. These cerebral signals are then digitized, so think about lifting their right arm becomes a “one” and thinking about lifting their left arm is a “zero”, for instance. The binary information is then transmitted over the internet from the first volunteer to the second volunteer, who are hooked up to their computers in a similar way.

The second PC translates the signal into flashing lights seen be the second volunteer. They don’t understand the message, but the electrodes measuring activity in their visual cortex feed their brain’s reaction to what they see into another computer that interprets the data to reveal whether the first person transmitted a zero or a one.

The research was carried out by scientists looking at the possibilities of brain-computer interfacing (BCI). This is where brain signals are captured and translated into commands that allow humans to control devices such as wheelchairs, robots or virtual reality environments. “This experiment came about through academic curiosity,” says Dr Christopher James of Southampton University’s Institute of Sound and Vibration Research.

James claims the experiment proves that brain-to-brain communication is possible. If so, it has the potential to help people with conditions such as “locked-in syndrome”. This is when someone can’t communicate – the patient is aware and awake, but unable to move or talk because neally all their voluntary muscles are paralyzed. And in the world of gaming, it could be a means to allow gamers to chat with each other.

The Green Olympic Games

The 2010 Vancouver Olympics is going green and raising the figurative bar with high flying sustainability initiatives. The upcoming Olympics set to begin on 12 February 2010 will be the greenest games in history. The Olympic committee, along with many national organizations and corporate sponsors, has been planning on launching an eco-friendly games for years. The focus being that sustainability is important to all.

In the case of these Winter Olympic games set in Whistler, Vancouver sustainability means managing the environment, the social and economical impacts, and the opportunities of the games so as to produce lasting benefits both locally and globally. The Olympic committee recognizes that local and global sustainability challenges and opportunities can be met, and that to do this we must look back at the past and forward into the future. Here are some unique ways the dedicated committees are making a positive difference.

The move towards a green Olympics started by thinking sustainability. The committee expanded their education and programs in this area. Sustainability videos were launched on their website to raise awareness and state their plans. An entertaining animated video to educate and raise environmental awareness for all was created for the Olympics and will be played at the event. It is currently available on the official Olympic website. (www.vancouver2010.com)

The committees are working with indigenous peoples of British Columbia to create partnerships that will promote their legacies and honor their territories and culture. Sustainability has been integrated into the committee’s operation by putting goals in motion and actively tracking the measures of their performance.
Goals set forth are:

Advanced social inclusion by reaching out to inner city residents and businesses, as well as the aboriginal people and others who don’t usually participate.

Going Greener by incorporating LEED standards in the venue’s design and construction. These sustainable venues consist of smart site selection and smaller footprints which lead to conserving and saving of all local vegetation and wildlife. Wood waste reuse was implemented. Solar panels are installed and Salvaged Beetle wood was used to create all roofs. Rainwater capture and waste heat reuse powers the oval. The floors in the Village buildings are heated from below, which minimizes dust particles in the air. The village buildings have an in-slab hydraulic system that will be used in heating and cooling all of the buildings.

Convening carbon neutral games by taking the initiative to forecasting, reducing, offsetting and reporting on the its carbon imprint. Visitors are encouraged to calculate their carbon emissions and offset their consumption by paying an offset fee that goes to clean technology programs. The committee has addressed the transportation issue by offering 20 hydrogen powered buses. A walk, hike or bike campaign is being launched. All are an important part in reducing carbons.

The Winter Games is committed to KNOW, REDUCE, OFFSET, and INSPIRE.

Doing more with less is the best. We all must do our parts. One company doing their part is Coca Cola. They have launched an ambitious Eco-Friendly campaign to produce zero waste during the Vancouver Games. The effort is years in the making, beginning with their recycle program in the 2000 Olympic games. Since then, the company has been a leader in being environmentally friendly.

Here is how Coca Cola is promoting sustainability at the Vancouver games:

  1. All coolers will use ekofresh technology which eliminates greenhouse gas emissions.
  2. They will use diesel hybrid vehicles and electric carts for delivery.
  3. Tables and chairs in the athletes village will be made of salvaged wood from the pine beetle epidemic.
  4. All staff uniforms will be made of recyclable bottles. This includes t-shirts, bags, and sweat suits.
  5. All employees carbon offset fees will be paid.
  6. All materials used will be made recyclable materials.
  7. Recycle bins will be provided everywhere.
  8. Green Teams have been created to clean up and promote sustainability.

New Program Offers FREE Online Training

A new training program is being offered through the Eastern Technology Council that allows employees from qualified businesses to take information technology and professional courses online at no cost. The course list includes basic computer skills, information technology and healthcare training. The cost-saving element of the program is immense. I’ve seen tuition for equivalent technology courses costing as much as $700 to $1,000, said Dianne Strunk, CEO of the Eastern Technology Council.
MVM Associates, an organization that identifies and secures tax credits, grants and other financial incentives for private companies, is handling the programs application process. Were excited to help companies find a way to meet growing training obligations, she said. Students taking the online courses may also take advantage of KnowledgePools online mentoring service. The firms mentors will provide real-time, anytime help. For more information, call 215-540-8463, or visit www.mvmgrants.com.

Eligibility Requirements

Employers must:

Maintain a Pennsylvania presence.
Be approved for the special e-Learning Grant via an application process.
Employers should contact MVM Associates for the application procedure. For IT training, all companies, except those noted below, are eligible. Point of sale retail businesses, employment agencies, government, education, training vendors and non-profit entities are not eligible. However, if your business does fall into these categories, please contact us about other available alternatives.Employers must verify that employees who participate in the training:
Earn at least 150 percent of the minimum wage.
Are permanent full-time employees receiving full- time benefits.
Are verifiable residents of Pennsylvania and are employed in Pennsylvania.
For IT training, are information technology professionals.
The following courses are being offered:

Desktop Computing

Microsoft Office XP
Microsoft Office 2000
Microsoft Office 95
Microsoft Office 97 and 98
Lotus Notes
Novell
SAP
Business and Professional Development:

Accounting
Finance and Business Law
Communication
Customer Service and Consulting
Financial Services
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Strategic Planning and Project Management Curriculum Team Building
Information Technology:

CISCO
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Java 2.0, HTML, XML and XHTML
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Windows 2000
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Visual Studio 6.0
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Visual Basic 5 and 6
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Oracle 8, 8i and 9i
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Oracle Business Intelligence
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Oracle Designer and Developer/2000 release Curriculum
C/C++ Programming and Systems Analysis
UNIX Administration and Programming