Tag Archives: PacBio RS

PacBio Forecast 2015

ID-10081802As already predicted, it is not only Illumina who communicates innovations for their NGS portfolio. Here you can read about the implementations Pacific Biosciences plans this this. I think the good news for many users of PacBio machines is, that they do not talk about new instruments, but improvments that affect already installed machines (GenomeWeb):

  • PacBio plans to improve the sequencing chemistry, including the active loading of single polymerase enzymes onto the chip
  • PacBio plans to improve the workflows for an easier and faster handling of samples
  • PacBio plans to improve bioinformatics for faster de novo genome assemblies & better analysis of full-length HLA analysis

With this changes PacBio wants to extend the data output to more than 4 gigabases / SMRT cell and increase the average read lengths to 15-20 kbp.

Read more about it here.

I still wonder if there will be news from PacBio this year about a new system? Maybe a benchtop like everyone has?

I will keep you updated!

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.

Whose Genome Has Been Sequenced? Belgica antarctica

de-novo-sequencingExtreme conditions require extreme actions. And this is what the midge Belgica antarctica has done. The midge lives exclusively in the Antarctic and in order to survive shrinked its genome to the smallest possible size. As of today, this is the smallest insect genome that has been sequenced.

Kelley et. al. now sequenced the genome of Belgica antarctica with the aim to learn more about how insects in general can adapt to the most extreme conditions.

What was sequenced?

Two fourth instar larva (Belgica antarctica) collected near Palmer Station, Antarctica.

Sequencing strategy: Whole genome sequencing & RNA-sequencing

  1. Libraries & Sequencing: 1 channel 2x 100 bp Illumina HiSeq 2000 (SG library (400 bp insert)) and one SMRT-cell of a 10 kb fragment library on PacBio RSII (P4 DNA Polymerase)
  2. Data output: 92 M paired-end reads from the shotgun sequencing with Illumina. These resulted in 5,422 contigs. Using the paired-end RNA-Seq data the number of contigs has been reduced to 5,064. Genome coverage with Illumina sequencing ~ 100x.
  3. Results: The total genome is ~ 99 Mbp.

For the PacBio sequencing a second larvae was used. But due to the low input of genomic DNA the PacBio data yielded only in a modest improvement in assembly. This underlines the need of a long-read sequencing technology with low input DNA material.

The de novo sequencing of the midge Belgica antarctica revealed that the smalll genome size is achieved by a reduction in repeats, TEs and intron size.

Read the complete publication here.

Whose Genome Has Been Sequenced? – Recent posts:

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.

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.

All you need to know about NGS

During my research through the web for news in the area of NGS I found this great practical course:

The EMBL-EBI offers a 15h online course that should help every newby and everyone who needs some refreshing information in NGS. And since we have several users of this blog that are new to NGS I thought it would be worth to share this course.

This course is divided into several subunits so everyone can learn in his own speed and in alignment with other tasks that are on everyone’s desk.

 

The key learnings are:

  • Understand some principles behind NGS
  • Know the challenges created by NGS
  • Know how to submit and retrieve NGS data to and from databases
  • Understand the uses of NGS data in: Whole genome assembly; Gene expression analysis; Genome annotation; Gene regulation analysis; Variation studies

Thank you EMBL-EBI for this great summary!

Possibility of Ideal Intestinal Remedy

I’m not able to keep intestinal condition without remedy which is prepared by Lactobacillus, Bifidobacterium, Lactococcus, and others. Eating yogurt is also okay for this purpose, ad personam I prefer to take these bacterial tablets and believe more effects. However, many people know these effects of current intestinal remedies are mild not fast-acting properties.

Several reports mentioned that intestinal bacterial flora and its regulation were not simple. It is starting discussion that natural immunity may regulate intestinal flora; e.g. antibacterial peptide α-defensins which is secreted from paneth cells on small-intestinal epithelium could regulate flora distribution (Salzman et al., 2010; Matsuda et al., 2011). By contrary, very simple strategy is reported as following: Clostridium difficile brings bad diarrhea that was resistant to antibiotic. Van Nood and co-workers (2013) injected healthy person’s feces into patient’s guts, and its curative effect was so good surprisingly, but we cannot call it a remedy!

Two NGS platforms, GS FLX/Junior and MiSeq, can perform distributional analysis via deep sequencing of 16S-rRNA amplicons. But it is still difficult for both platforms to do metagenome assembling for getting whole gene information in flora, because their read length is not long enough to make reliable contigs without chimeras between different bacteria one another. Therefore I strongly expect that super long read platforms including PacBio RS series and coming nano-pore technologies will break current limitations and will contribute to develop ideal intestinal remedy for my instable stomach.

News From The World Of NGS

Pacific Bioscience’s instrument PacBio RS is known as the third generation sequencing technology. And again they proof their innovative character. During the course of the last couple of months two new chemistry packages have been released (XL & P4). And during the last couple of days two even more interesting news were spread:

1. Roche and PacBio’s signed an agreement for codeveloping diagnostic products for the PacBio RS instrument (Genome Web). From my point of view this is a huge signal. Roche, as an experienced player in the NGS market with its own sequencing instruments see’s a lot of potential in the SMRT technology. So the PacBio RS system obviously got out of the teething phase and will increase its importance in the NGS business in the coming months.

2. And also New England Biolabs a big player in the area of enzyme production, proteomics and drug discovery is using the PacBio RS to study bacterial methylomes and work on new reagents for 5-mc detection. And the CSO from NEB highlights that they have choosen “the PacBio system to study bacterial methylomes because of a unique feature of SMRT sequencing that enables the detection of base modifications through the system’s kinetics”. (Genome Web)

Beside the great news for Pacific Biosciences als Life Technologies or better Thermo Fisher signed a great deal with the Chinese Dx firm iGenomics to install 32 Ion Proton sequences in 2013 (Genome Web).

And clearly all developments focus on Molecular Diagnotics and Clinical Diagnostics. And to add the missing link in this news update also Illumina recently announced that they partner with G3 to identify novel biomarkers and pathways in cardiovascular disease.

Summary from 4th Next Generation Sequencing Congress 2012 – Part 2

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: —

Kevin-Corcoran-Pacific-Biosciences_Seite_16

 

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: —

Kevin-Corcoran-Pacific-Biosciences_Seite_52

 

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: —

Kevin-Corcoran-Pacific-Biosciences_Seite_56

 

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

Further Improvements of PacBio Technology

Recently, we have reported on the Studies of the Broad Institute, showing that the PacBio RS system was able to outdo MiSeq sequencing regarding validation of SNP analysis. Now Pacific Biosciences have taken another important step to further improve their product.

Pacific Biosciences have now launched a new Sample Loading Device for the PacBio RS, called MagBead Station.  As  Michael Hunkapiller, Ph.D. President and Chief Executive Officer of Pacific Biosciences told in their press release, they expect that with the new device, customers will  “be able to generate 10 kilobase-sized libraries using as little as one microgram of sample, a five to 10-fold improvement from where we were just a few months ago”. Also, because the new process is more robust, they expect that sequencing results will have higher overall consistency, allowing to run experiments also on challenging samples.

First experiences of early-access-customers seem to underline these expections:

As Patrick Hurban of Expression Analysis told InSequence, the new loading device allowed them to recover sequences also for “difficult” samples: “we’re much more confident on a sample-by-sample basis that we will be able to get good sequence”, he said. Also, they could confirm that the amount of library that needs to be loaded is now significantly lower. The new loading process also seems to favor longer DNA fragments over shorter ones, excluding short contaminating DNA fragments. This results in a greater percentage of long reads in a run. Also, the loading process now seems to work as efficiently for the large insert libraries as it does for the smaller insert libraries.

With the new loading device, about 50-60 % of the ZMWs are now active after loading. This is a great improvement compared to 30-45 % of active ZMWs before the upgrade.

When PacBio started on the market, I was impressed by the sophisticated new technology. However, the results of the first projects were rather disappointing. The new loading device now seems to greatly improve the sample loading step. However, the high error rates still remain a challenge, with about 15% for the time being. Pac Bio will need to solve those issues if they want to be successful on the market in the long run. However, it seems that by and by, PacBio is overcoming  its “childhood diseases”.