Tag Archives: Next generation sequencing

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!

Don’t forget the controls!

Almost every day new data about the composition of microbiomes are published. Many of these studies analyse the human microbiome, but also environmental samples.

Today we have the ability to sequence microbiomes in much more depth than a couple of years ago. Looking deeper sheds light on an important point: Contamination! In the very interesting publication of Salter et al. they could show that contaminating DNA is present in DNA extraction kits and other lab reagents.

The researchers sent dilutions of pure cultures of Salmonella bongori to three different institutes for DNA extraction and PCR, followed by sequencing on Illumina MiSeq. While S. bongori was the only organism identified in the undiluted samples, contaminating bacteria increased in relative abundance with higher degrees of dilution, and finally became dominant after the fifth dilution.

They did a similar analysis performing shotgun metagenomics of a pure S. bongori culture. This time, they used four different DNA extraction kits. Again, they saw that contamination increased with the degree of dilution, with contamination being the predominant feature after the fourth dilution. Also, they could show that each kit gave a different bacterial profile.

They also report on a study on the nasopharyngeal microbiota of children, analyzed over 2 years. They could show that using 4 different DNA extraction kits over time led to the false conclusion that differences in the microbial spectrum were associated with age. When DNA extraction was repeated on original samples using a different kit lot, the OTUs previously identified as contaminants were no longer detected.

In conclusion, contamination affected both 16S and metagenomic shotgun sequencing projects and was especially critical for samples with low biomass. Salter et al. present a list of potential contaminating organisms, as well as recommendations on how to cope with this problem. One recommendation is very obvious, and very effective: use negative controls!

Altogether, we should be very careful in planning our experiments in order to deliver results instead of artefacts. Especially, we need to be very careful when interpreting the data!

Quality Before Quantity?

The  “$1.000 genome” is to my knowledge the buzzword everyone knows when thinking about Next Generation Sequencing.

And quite often I ask myself: What will be the future of NGS? Whole genome sequencing of everyone and everything?

I am confident that this is part of Illumina’s  strategy for their new HiSeq X Ten instruments – at least for humans.

Contrary there is still all the data that needs to be analysed. And an interview with Lex Nederbragt highlights that data analysis is still a bottleneck. Also the latest report from Markets&Markets for whole exome sequencing predicts a strong growth in targetd sequencing. They estimate a growth for whole exome sequencing  from $326.6 M in 2013 up to $884.1 Million by 2018.

So will quality, like sequencing distinct regions outcompete the $1,000 genome? What are your thoughts about that?

Data analysis – still a bottleneck!

With the many NGS machines around in the field, we daily produce tremendous amounts of sequencing data. However, at the end of the day, all the data have to be analyzed and interpreted. In many cases, this step is still a bottleneck.

Please check the video below which is an interview with Lex Nederbragt, Bioinformatician at the Norwegian High-Throughput Sequencing Centre in Oslo, on this topic. He discusses the fact that the analysis tools which are available do not fully fulfill the needs of the researchers. In this context, he also discusses the use of open source and commercial software tools.

Lex Nederbragt discussing software bottlenecks and lack of flexible reference genomes from NGS Perspectives on Vimeo.

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.

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 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…

paper_02

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.

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!