First Oxford Nanopore MinIon data available: Is this the end of PacBio?

Nanopore SequencingResearchers from the University of Birmingham in the UK last week publicly released data they generated with Oxford Nanopore Technologies’ MinIon nanopore sequencer, the first group to do so since the company started its early access program this spring (see In Sequence report).

The sequence is derived from a Pseudomonas aeruginosa genome and is a single 8.5 kilobase read. It was posted by Nick Loman from the institute of Microbiology and Infection at the University of Birmingham. It was possible to identify the serotype O6. The sequence can be found here. It is of low quality with 71% identity of the spanned region.

Konrad Paszkiewicz, director of the Wellcome Trust Biomedical Informatics Hub and head of the sequencing service at Exeter, has been writing about the group’s experience on the Exeter Sequencing Service’s blog. “Even at this stage, this platform has the potential to steal large chunks out of the market from the likes of PacBio,” Paszkiewicz said.

We will have to wait for more data until we see how useful the technique will be and how the technique is able to compete against other Nanopore sequencers e.g. the device of Genia that was recently acquired by Roche.

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Improvement of PacBio ZMW loading procedure by DNA Origami?

Since the launch of the PacBio system in 2011, there has been a constant development and improvement of the methods involved (e.g. former posts here).

OrigamiStar-BlackPen.pngHowever, efficient loading of the Zero-Mode Waveguides (ZMWs) with polymerase molecules still remains a challenge. The ZMWs are tiny wells in which the actual sequencing reactions take place. Each SMRT cell consists of 150,000 ZMWs. However, with current methods, only about 1/3 of the ZMWs is actually useable after loading. The polymerase molecules are loaded onto the ZMWs by simple diffusion – resulting in ZMWs which carry one, more than one, or no polymerase molecule. As a consequence, each SMRT cell typically generates only approx. 50,000 reads per run.

A group of researchers from the Technical University of Braunschweig, Germany, has now used “DNA Origami” in order to efficiently place molecules into ZMWs.

DNA origami is a fascinating technique which uses the unique properties of DNA in order to create nanostructures by “folding” DNA into the required shapes. A ground-breaking article on DNA origami has been written by Paul Rothemund in 2006.

The researchers from Braunschweig have now created “nanoadapters” which exactly fit the size of the ZMWs. As a consequence, there cannot be more than one molecule in a ZMW. The nanoadapters carry a fluorescent dye on top and biotin molecules on the bottom side. These biotin molecules serve in fixing the nanoadapters to the bottom of the ZMW via neutravidin. In principle, the fluorescent dye could be replaced by a polymerase molecule. This approach greatly increased the loading efficiency to approx. 60 percent.

However, according to InSequence, the research group did not co-operate with PacBio for this project. In parallel, PacBio is working on other methods to increase the loading efficiency of their SMRT cells. But I am sure that there will be (and has to be) an improvement soon- no matter by which methods.
OrigamiStar-BlackPen” by Aldaron, a.k.a. Aldaron. – From JillsArt, posted with permission. Licensed under Attribution via Wikimedia Commons.

Exome Sequencing At A Glance

Selective characterisation of the genome’s complete coding region

In humans, only 1-2 % of the genome is protein coding, the so-called exome. Exome sequencing is favoured over whole genome sequencing due to costs, efficiency and the easier interpretability of a much lower data volume compared to whole genome sequencing. It gains more and more clinical relevance in the determination of rare diseases as well as for cancer research and diagnostics. Furthermore, it’s a very important screening tool for genetic variations e. g. involved in mental disorders such as schizophrenia and is therefore increasingly used as one genomic application in drug discovery. Exome analyses are frequently conducted as trio analyses with one patient plus healthy parents, who serve as controls to filter out benign variants. They are not only performed on behalf of companies or academic research organisations, but also gain more importance in diagnostic applications for individuals.

The most common technologies for exome analysis are based on in-solution hybridisation. They use a protocol that first generates a whole genome library, and then enriches the exome portion of the genome. The well-established kits for this kind of analysis are from NimbleGen, Agilent and Illumina. The exome enriched DNA is then primarily sequenced with Next Generation Sequencing systems from Ilumina, like Illumina HiSeq. This approach is typically selected for projects with large sample numbers. One limitation is the incomplete coverage for some genetic loci. More consistent sequence coverage can be achieved by using a PCR based exome capture approach offered by Ion Torrent. This approach allows a very fast and a more uniform exome analysis ideal for small to mid-size sample numbers.

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Work where others make holidays !

Prof. Leonid Moroz from the University of Florida has become the first scientist to sequence the genome of fragile marine creatures on board a ship in real-time (see scientific computing world).

copaseticBecause of the difficulties of storing or shipping their genetic material, it has hitherto been difficult to sequence the genomes of marine species. However, researchers at the University of Florida have got round this problem by deploying a fully-equipped genomic laboratory aboard a ship called the Copasetic and sending the initial data via a satellite link to the University’s new HiPerGator supercomputer.

Aboard the Copasetic in early February and later in March-April, Professor Leonid Moroz, from the University of Florida, and his team where able to perform transcriptome sequencing of 22 organisms, among them rare comb jellies.

The first results of the sequencing at sea were presented at the international conference, Advances in Genome Biology and Technology, held at Marco Island, Florida in February.

Next Generation Sequencing Market Trends

paper_02The GEN report by Enal Razvi, Ph.D. provides an overview of the current NGS field in terms of application areas and utilization patterns.

Some findings of the report:

    • The exponential growth of NGS-focused publications illustrates the expansion of NGS and its penetration info research.
    • 49% of next generation sequencing methods are used for basic research.
    • 29% of researchers are using NGS for comparative genome sequencing
    • 38% of research efforts are studying somatic mutation
      33% are studying mRNA expression via RNA-Seq

Is China breaking the dominance of Illumina?

BIGIS-4 is the name of an independently developed next generation sequencer made in China. The sequencer shall challenge the dominance of Illumina. On 18 April, scientists from the Beijing Institute of Genomics (BIG) of the Chinese Academy of Sciences and partner company Zixin Pharmaceutical Industrial Co Ltd demonstrated their BIGIS-4 sequencing machine in Changchun, Jilin province.

The Chinese machine has a longer read length than dominant sequencers like those made by Illumina in the US. Its manufacturing cost will be one third cheaper than imported machines, and operation costs about one fifth lower, according to Yu Jun of BIG, chief scientist of the project. Yu was also a co-founder of Shenzhen-based BGI, a spin-off of BIG and now the world’s largest sequencing service provider.

Yu’s sequencer differs from Illumina’s in that the fluorescent tag is cleaved from the newly synthesised DNA as it is incorporated, so that the reading speed is much quicker. This is similar to the pyrosequencing technology employed by Roche Diagnostics’ subsidiary 454 Life Sciences.

A publication about the complete genome sequencing and assembly of a Glaciecola mesophila spec. with BIGIS-4 is published here.

Nanopore MinION to be tested and evaluated in Sweden

There are many researchers and service providers who are talking about the MinION from Nanopore on a regular basis. Great things are expected from this innovative sequencing technology.

As previously mentioned, Oxford Nanopore has shipped some MinIon devices to early access users to receive some more data. As one of the few laboratories in Scandinavia, the Department of Animal Breeding and Genetics at the Swedish University of Agricultural Sciences has been chosen to test one of the latest sequencing technologies from Oxford Nanopore. By the size of the sequencer it is possible to bring the sequencer in the field.

Previously the department has tested leading NGS technologies in research related to the genes involved in the immune system of horses. Now the Nanopore technology will be used, compared and evaluated against the existing well-proven sequencing technologies.

I guess it is just to wait and see what the MinION will bring, if it will come up to expectations or not.

New method: single cell genome sequencing of malaria parasites

Single cell genomics provides new insight into the biology of Malaria parasites (Plasmodium vivax and Plasmodium falciparum), including their virulence and levels of drug resistance to improve treatment and control of the disease. 

mosquitoThe new method for isolating and genome sequencing an individual malaria parasite cell will allow scientists to improve their ability to identify the multiple types of malaria parasites infecting patients and lead to ways to design drugs and vaccines to tackle this major global killer.
Malaria parasite infections are complex and often contain multiple different parasite genotypes and even different parasite species. So when researchers take a blood sample from a malaria infected patient and look at the parasite DNA within they end up with a complex mixture that is difficult to interpret.

“Current sequencing techniques really limit our understanding of malaria parasite biology” says Ian Cheeseman, Ph.D., who led this project. “It’s like trying to understand human genetics by making DNA from everyone in a village at once. The data is all jumbled up – what we really want is information from individuals.”

To achieve a better understanding of malaria parasites – single celled organisms that infect red blood cells – the project team developed a novel method for isolating an individual parasite cell and sequencing its genome. Single cell genomics allows the separation and isolation of cells to extract and sequence individual parasite DNA and determine any differences between the parasites within an infection..

“One of the real challenges was learning how to cope with the tiny amounts of DNA involved. In a single cell we have a thousand million millionth of a gram of DNA. It took a lot of effort before we developed a method where we simply didn’t lose this,” said Nair, the first author on the work.

Their method is set to change how researchers think about infections. “One of the major surprises we found when we started looking at individual parasites instead of whole infections was the level of variation in drug resistance genes. The patterns we saw suggested that different parasites within a single malaria infection would react very differently to drug treatment” said Nair.

Unfortunately the new method is currently too expensive and demanding for routine use in the clinic, as the technology matures the applications for understanding malaria biology are vast.

___________________________

The findings are revealed in a study by researchers at the Texas Biomedical Research Institute and published recently in the journal Genome Research.

The work is funded by the Texas Biomedical Forum, National Institutes of Health, a Cowles Postdoctoral Training Fellowship and the Wellcome Trust and was led by Texas Biomed’s Cheeseman with collaborators at the University of Texas Health Science Center San Antonio, Case Western Reserve University, the Cleveland Clinic Lerner Research Institute, the Shoklo Malaria Research Unit, Thailand, and the Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Malawi.

Get further details here.

New kids on the show – who will be the winner?

Next Generation Sequencing is still a quite young market. Therefore we face the same situation every year: there is a lot of innovation going on in regard to new technologies, new instruments and other inventions. Amongst all these innovations GenomeWeb picked out the new platforms and asked in a survey about the expectancies in the market.

Instruments that were part of this survey are (at least in one of several questions):

  • Oxford Nanopore’s MinIon
  • Illumina’s X-Ten
  • Illuminas NextSeq 500
  • QIAGEN’s GeneReader
  • Life Technologies’ Ion Torrent PGM
  • Illumina HiSeq

Here are some of the results:

  • 35% of the participants say that the MinIon has the greatest impact on the sequencing community
  • 30% of the participtants will purchase most likely the NextSeq 500
  • Illumina HiSeq / MiSeq outperform the Ion Torrent Proton / PGM in data accuracy and throughput, the Ion Torrent instruments are better in respect of run time and instrument price

Read the complete survey here.

NGS Applications – get an insight…

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You want to know more about projects where your research colleagues used next generation sequencing?

Check out the Nature Reviews overview of interesting publications releated to different applications of next generation sequencing.