Tag Archives: Single molecule sequencing

PacBio launches new chemistry and software

In a press release Pacific Biosciences announced the latest enhancement for the PacBio RS II single molecule DNA sequencer. The latest release of the polymerase 6 and chemistry 4 (P6 – C4) version in combination with improved software enhances the performance and output of the platform by 45%. The average read length is now 10,000 – 15,000 bases and up to 40,000 bases for the longest reads. Depending on the nature of the DNA a single SMRT cell will deliver 500 million to 1 billion bases.

The new chemistry will replace the current P5 – C3 chemistry and is recommended for all SMRT sequencing applications.

This new release also includes improvements to the SMRT Analysis software suite for long amplicon analysis and the Iso-Seq™ method. Together with chemistry enhancements, these advances boost accuracy, speed up analysis, and support sequencing of multiplexed amplicons of different sizes.

Survey result: What do you think about Nanopore sequencing?

Earlier this year Oxford Nanopore Technologies presented their solution for Next Generation Sequencing: the MinIon & GridIon instruments outranges the current available techniques like Illumina or Roche systems by read length, hands on time and pricing. But since the technology is not launched yet, we don’t know if these specs are realistic.

This is why we asked you about your opinion in our latest poll (Nanopore sequencing from Oxford Nanopore Technologies sounds really fascinating. What is your opinion regarding this technology?). More than 50 voters took part in this survey and 42% share my opinon: “I prefer to wait and check out the real system before judging it”.

“Paper doesn’t blush” is what 15% think of this announcement – like every other company the first presentation needs to be spectacular, but let’s see what happens when the instrument is really on the market.

And still some of you are convinced that this will change a lot in the NGS market – and I agree it would be great if it turns out to be true.

Some of you haven’t heard about this technology – so if you are interested to learn more about it you might start by reading our recent blog post about it.

Thanks again to all you participated in our voting and please have a look at our new poll.

Focus on FLX+ ?

In the last weeks we were continuously following the hostile bid from Roche for Illumina. Now, after the shareholder meeting, Roche didn’t extend the offer and let Illumina from the hook – for the moment?
However, this could also be good news. From my point of view there is a wide range of applications that work best with the FLX technology. In order to be competitive with all other NGS technologies Roche needs to invest more in FLX+. After launching the upgrade last year they faced continuously problems with the performance of the technique as highlighted for example in an article in InSequence last week.

I think, because the NGS future of Roche is again more focussed on FLX+, they will work even harder to get the long reads up an running on every GS FLX site – maybe a FLX+ upgrade for the GS Junior might be possible soon.

Of course it also might be that they are looking for an alternative for Illumina as highlighted by Julia Karow and Monica Heger. However, all possiblities discussed in this article – a cooperation with Life (Ion Torrent), an acquisition of Oxford Nanopore technologies or the development of a brand new technique might not be as scary for the FLX technology as Illumina because all technologies are “very early in the commercialization”.

How Small Can Next Generation Sequencing Devices Get?

Scarcely any biotech blog and bulletin is not reporting about Oxford Nanopore Technologies MinIon & GridIon.

Truly, the specs of the new instruments sound fantastic: read length up to 100 kbp, raw read error rate ~1%, no sample preparation necessary (at least with blood samples), RNA can be sequenced directly, costs per base comparable to competitors, only 900$ for the MinIon device and data production in the range of 20-400 bases / second / pore (GenomeWeb).

And it goes without saying that these new instruments cause a lot of fuss in the Next Generation Community: shares from competitors are down up to 6 percent, and every one discusses fictive scenarios that might now be possible.

I am also really excited about this news and it still sound unbelievable that Next Gen Sequencing devices can be so small. But I also have my doubts  and agree with the following statements:

“… If only 75% of what they claimed is true it would be impressive.” (Pathogenomics)

“…that new DNA sequencers, like everything else in life, often look better when they’re vapourware.”(Forbes)

What is definitely true is that you really have to read also the small prints at the bottom of the page. You cannot, for example, use the USB-Stick-like device “MinIon” to sequence the complete genome in 15 minutes. You would need to run 20 GridIon instruments or “nodes” in parallel to achieve this turnaround time with a coverage of 50x.

So it is definitely fascinating what might be possible with the MinIon – just think about the point-of-care market, where we would need small, disposable devices to work on site. But still you should be able to interpret the data and I look forward to learning how they will solve this issue. And furthermore I am even more curious what kind of instruments we will be using in 5 years: Smaller or faster ones or something completely different? What do you think?

Oxford Nanopore Technologies Ltd.

Future of Strobe Sequencing with PacBio RS

Strobe sequencing is one of the three sequencing protocols of the PacBio RS that researchers have been waiting for some time. By switching the data acquisition on and off, the machine sequences stretches of DNA in bursts. On periods generate the so called strobe subreads while off periods determine the distance between individual subreads. The most important application of strobe sequencing is the improvement of de novo assemblies by scaffolding contigs. 

However, recently, PacBio’s CEO Hugh Martin announced that the company will no longer focus on further development of this technology. As the PacBio RS chemistry upgrade which is planned for Q4 will deliver average read length of 2700 bp with 5 % of reads > 5100 bp, strobe sequencing is getting more and more obsolete.

The performance of the protocol still is a challenge in praxis as early-access customers reported in May in Genomeweb. This is in accordance with our experience. First, sequencing does only generate few thousands of reads per SMRT cell, and second, many reads are observed having only one or two subreads.

Epigenetic Studies by direct Single-Molecule Sequencing?

Next generation sequencing allows researchers to generate high resolution maps of epigenetic modifications for whole genomes (epigenome). Whole genome methylation pattern has been determined e.g. for A. thaliana by sequencing bisulfite treated DNA with Illumina technology (Lister et al., 2008 and Cokus et al., 2008).

In the near future epigenetic studies may be possible without bisulfite treatment by single-molecule real-time sequencing (SMRT). The first proof of concept study promises direct detection of modified nucleotides as well as discrimination between different types of modifications (Flusberg et al., 2010).