NGS Expert Blog

The Genomics Virtual Lab (GVL) project – using the computing resources from the NeCTAR Research Cloud – is an Australian Government project conducted as part of the “Super Science” initiative. It is developing infrastructure supporting genome informatics research.

Their Galaxy-based NGS and HTS tutorials are really excellent:

• RNA-seq tutorial
• Variant detection tutorial
• Variant detection (advanced) tutorial
• Genome assembly tutorial

You will love the precise explanations, the hands-on demonstration and the additional material like screenshots and in-depth information!

Opinions differ as to whether next generation sequencing is already mature enough to be a useful tool in diagnostic routine.

Below you can find an interview of the cardiologists from the university of Heidelberg about their studies to integrate next generation sequencing into a diagnostic tool. Therefore they do collaborate with Siemens to receive best possible results that can be used by the doctors in the same way as current reports from other technologies.

Dear all,

Here is my second summary from 4th NGS Congress at London Heathrow end of 2012. It will bring to you some (hopefully) interesting new facts about sequencing with PacBio RS - the second long read technology present in the actual markets and also the only system delivering reads even longer than 10,000 bp…

Kevin Corcoran, Senior Vice President at Pacific Biosciences held an interesting and very nice talk about the most recent developments for the PacBio RS system. He also showed some nice detailed road maps about future aims and plans. One important thing actual to be mentioned is the launch of the new “XL Chemistry” – while still “C2 Chemistry” may be used as well. The other very interesting story is about “Stage Start” a new feature enabling a parallel start of all sequencing detection similar to the well-known “hot start” technology for PCR. Such detection of sequences better will start from a defined position for most of the libraries than starting from somewhere in the middle. Last but not least, I’m very keen to learn how the future “Photo Protected DNA Polymerases” may further develop – an idea being really very, very next-next-generation…

First of all I can summarize that applying “XL Chemistry” looks really interesting and this being true also in terms of Eurofins MWG Operon de novo sequencing and assembly focus.  This new feature of the PacBio RS machine may also open some new doors to other types of applications, while in general the need for extrem high data coverages may be reduced in parallel.

Currently “C2 Chemistry” is on the machine and running a 90 min video may deliver you about 20-50,000 reads and data outputs of 30-50 Mb – of course higher yields may be possible for “ideal” DNA samples. The average read length is about 3,000 bp (!), while the 95% percentile is about 8,000bp. With the new ”XL Chemistry” we got an average yield of about 40,000 reads per SMRT cell with an average read length of about 4,000bp (+30%). Overall, we are very pleased with these first results, especially since we see some good potential to further increase data yields using the new software pipeline started in parallel (Hierarchical Genome Assembly Process and Quiver).

— See picture 1: —

It is also important to mention two different ways of “How-to-deal” with the XL Chemistry. 1) ”XL chemistry for Polymerase binding”, but “C2 chemistry for sequencing”. This allows for longer reads at the same quality (currently we still do have a single error rate of 10% to 20 %, average maybe 15%). 2) “XL chemistry for Polymerase binding” AND ”XL chemistry for sequencing”. Such one can yield even longer reads, but unfortunately the error rate will also increase by a few %. Therefore this method is being recommended especially for de novo assembly or finishing genomes.

— See picture 2: —

Finally one real “next-next-gen” highlight was the presentation of a development at Pacific Biosciences scoping with the idea to protect the polymerase enzyme from being killed by the energy of the laser. A picture shows how this should work in principle - by setting in place a laser-light protecting sun-blocker - this story was really fascinating for me and I hope to see in future more than the very promising first data results …

— See picture 3: —

So over all Pacific BioSciences keeps also moving very fast in year 2013 and it will be very nice to see and learn how all these additional improvements and new features may  improve the overall data results of this fascinating very long read technology offering today real single reads longer than 10,000 bp.

Cheers now and see you on our next BLOG,
Axel

Adventitious viruses are a major safety concern in biological products. For a substance to be considered “free” of an adventitious agent, assays must demonstrate that a defined quantity of the biological product is negative for an agent at a defined level of sensitivity. In vivo animal testing, in vitro cell culture testing, transmission electron microscopy and molecular assays like quantitative PCR (qPCR) are the current gold standards for viral safety testing. However, if for example the cell substrate contains potential contaminating agents coming from a tumor derived cell line, then current standard methods need to be supplemented by using novel technologies.

Deep sequencing approaches via the next generation sequencing (NGS) techniques may be the method of choice. They allow the detection not only of known viruses but also of unknown viruses or viral subspecies at the detection limit of qPCR-based methods. On the Pathogen Safety Summit (Munich (Germany), November 27-28, 2012) the application of NGS testing approaches were introduced and intensely discussed. The application of NGS into routine testing of production cell banks is presently evaluated by several biological and vaccine producing companies.

Currently, NGS is used for initial characterisation of cell banks, but it iss expected that this new technology will become a standard method for adventitious agent testing soon. There are still challenges that need to be overcome with regard to bioinformatic analyses as well as to the speed of the technological development. Furthermore, also the biological relevance of the NGS data needs to be confirmed. In this regard the expectation is that with the ability to purify active viral particles and subject them to NGS analysis this problem can be overcome.

Btw: Eurofins Medigenomix offers the detection of adventitious viruses in biologicals and biotechnological products by next generation sequencing on platforms from Illumina and Roche 454.

With the advent of NGS came the promise of personalised medicine (PM) based on our unique genetic differences. The link below takes you to a seminar series from Harvard University which discusses the current status of NGS and its applications in a clinical setting.

There are many hurdles still to leap before PM is a mainstay of clinical diagnosis and treatment, reduction in cost, clinical grade databases, patient sceptisism and insurance implications to name but a few. However, we are at the begining of a revolution in clinical diagnostics and we cannot turn back now. The benefits of rapid diagnosis are obvious, provided we have the associated therapies to treat them. Otherwise it is just knowledge but without the power. There has been disappointingly slow progress in gene therapies and focus on this must also be maintained if the benefits of personalised medicine are to be realised. Furthermore,  in relation to a previous post – NGS and personalised medicine may also help characterise the complex relationship of disease and health, infection and immunological defense between ourselves and our personalised microbiome.

A recent study (Lancet Infect Dis. 2011 May;11(5):355-62. Epub 2011 Apr) exploring the transfer of the drug resistance gene NDM1 (New Delhi Metallo-Beta Lactamase-1) highlights the practical advatages of NGS.

In a realtively very short time the researchers in the paper were able to isolate the plasmids carring the the NDM-1 gene and sequence 12 plasmids to confirm that the transfer of the NDM-1 (drug resistance gene) amongst other species of bacteria. The implications are serious. Bacteria do not necessarily need to be challenged with a drug or be susceptable for long periods to develop resistance. The resistance can simply be transformed amongst adiverse microbial population. The use of NGS meant that all plasmids could be sequenced in one run to determine the previously unkown, location and the presence of the NDM-1 gene.

Conventional sequencing of over 160Kb of plasmids to try and locate the resisitance marker would have taken a great deal more effort. Thus, NGS is a key tool to track the spread of resisitance genes and hopefully enable researchers to develop prophelactic solutions. Potentially by sequencingf the genomes of all know phages- the natural viral enemy of bacteria. NGS provides another tool to hasten the end to the resistance wars.