Tag Archives: GS FLX

The Common Marmoset as a Model Organism for the Study of Drug Metabolism

marmosetSeveral non-human primates including Macaca mulatta and Macaca fascicularis are well known as experimental animals in the field of neuroscience, stem cell research, drug toxicology, and other applications. The common marmoset (Callithrix jacchus) is also a non-human primate and is suitable as experimental animal because of the small size and highfecundity.

For developing a drug metabolism model, our collaborators and Eurofins Genomics (2014) performed transcriptome analysis of the common marmoset using in parallel long-read technology (Roche GS FLX+) and short-read sequencing (Illumina HiSeq 2000). This parallel NGS approach resulted in both, the identification and the quantitative analysis of transcripts and thus giving insight into gene expression during drug metabolism. Finally we obtained rich information about genes involved in drug-metabolism including 18 cytochrome P450- and 4 flavin-containing monooxygenase -like (FMO) genes, and their tissue-specific expression patterns.

The results of this study are the foundation for future studies not limited to drug metabolism & pharmacokinetics.

Summary from 4th Next Generation Sequencing Congress 2012

Attending the 4th NGS Congress 2012 at London Heathrow I can give here some interesting new facts and information about latest NGS stories which are worth to be shared.

First of all let’s talk about “long read technology” – A Roche 454 talk has been given by Todd Arnold, Vice President R&D, Roche 454.  For Roche GS Junior a new software version 2.7, with  “improved well resolution results in better quality, more robust sequencing runs”  is now available.  As a matter of fact we can confirm these new data outputs while using on our own Junior platform with this update since a while.  Depending on your samples nature  a good part of all reads will be longer than 400 bp and up to 450-480 bp (still using the Titanium Chemistry). But the FLX+ technology is NOT available and also NOT planned for GS Junior – raising the question why,  no concret details or upgrade plans could be given for GS Junior at the London congress…

The real and major highlight about Roche 454 was the description of what we call now “FLX++” sequencing. A software update (2.8) being available now for all the GS FLX systems – together with  the “pimped chemsitry kits” – Roche 454 is offering real “1000bp” Sanger-like reads (as initially aimed at launch).  Some data outputs and slides were shown that demonstrate these new and longer read lengths and also higher data outputs (figure 1). All together that counts up to almost ~1Gb of sequencing data per full PPT run.

Fig 1: Todd Arnold Roche 454 Data Heathrow 2012

Being one of the early access users of the FLX++ upgrades and software version 2.8, we can in fact confirm that the new data outputs are excellent (again depending on the quality of DNA) – in fact one can reach even better results than shown by Roche at the 4th NGS congress in London Heathrow. Here is an example:

Fig 2: Eurofins MWG Operon data with Roche GS FLX++

Of course one may argue now – “that’s nothing compared to Illumina data outputs” – and you are right in terms of the pure data volumes! But the focus here is on long read applications like e.g. sequencing and de novo assembly. And for this kind of NGS application, a modal read length of 800-950 bp or above will tune the final data outputs treamendously. You won’t believe? We can share with you some nice new project data that we have delivered for a fungal de novo sequencing project (figure 2). We were able to deliver chromosome-size scaffolds of 8.3 Mb, 6.0 Mb, 4.3 Mb, 2.8 Mb, 2.4Mb, 2.1 Mb, … when using a long read FLX++ back-bone sequencing at  8x-12x only and combining this data with short read LJD sequencing on HiSeq at 2x 100 bp. The complete data set missed only about 0.5% of all genetic information, while remaining average gap lenght was about 240 bp.  We are actually very interested to learn how 2x 250 bp read length on MiSeq will further improve this excellent data results – one shot genome sequencing at it’s best.

Interested in this kind of project data? Please learn more about our fascinating de novo sequencing & assembly results at our next NGS roadshow in 2013 or send me an email for further discussion about this topic…

Sequencing than soaking in Hot Spring

There are many volcanoes and earthquakes in Japan, but it is not always a bad thing, they are also responsible for the many hot springs. Most Japanese people love soaking in a hot spring and they believe that this eliminates fatigue and improves health. Hot springs also had a great contribution to biotechnology via the heat resistant DNA polymerase from Thermus aquaticus (Taq) and its derivatives. Not only PCR, but also Sanger sequencing was accelerated by these heat resistant enzymes as we all know well.

Scientists have started to study the genome/transcriptome world in hot springs with NGS technologies. Murakami et al., peformed 16S-rRNA (Sanger sequencing) and meta-transcriptome analysis from small RNA (GS FLX sequencing) of groundwater (up to 1,000 m depth) from Yunohara hot spring, Japan. Their phylogenetic analysis using 16S rRNA showed the classification of 17 species including archaea and eubacteria.  There are only 2 or 3 dominant species in typical cases of other hot springs, but this one is rich in diversity. Furthermore, they found the very unique group “Archaeal Richmond Mine Acidophilic Nanoorganisms (ARMAN)” which is a small organism/cell with only 200 nm size! Their small RNA analysis identified 64,194 (20,057 nonredundant) cDNA sequences, and they found several novel non coding RNAs which have a very stable secondary structure.

Therefore, hot springs may still be gold mines for useful genes and important biological knowledge of unknown underground ecosystems.

 

 

Survey Result:
Applications Using Roche GS Technology Providing Read Length <700 bp

We asked, for which kind of application do you use the Roche GS FLX+, the GS FLX Titanium or GS Junior sequencing technology providing read length of up to 700 bp? 36 people answered the poll:

 

 

 

 

 

 

 

 

 

Please find on the right hand side our new poll and add your voice!

How dirty is your office?

16S rRNA sequencing of samples from 54 office-common surfaces in 3 different cities (New York, San Franscisco, Tuscon) revealed that offices of men are dirtier than these of women and the offices in San Francisco are the cleanest among the three cities. This is part of the results from Hewitt et al. published in PLoS ONE just recently. Overall they found “more than 500 bacterial genera from 20 different divisions” (Hewitt et al.) whereas most could be found on chairs and phones (see graph). But interestingly the bacterial population from Tuscon was significant different to the one from San Francisco and NewYork although the distance between Tuscon and San Francisco is smaller. From my point of view this is a great study showing that distribution is not as obvious as we think and that we haven’t revealed every secret on earth yet.

Spring Special – Amplicon Sequencing


  • Would you like to detect variances down to a frequency of 0.1% in your PCR sample?
  • Are you interested in a short turn around time?
  • You need primers for your amplicons?

 

Amplicon sequencing is still under discussion. Which technology is most suitable? In one of our latest blog posts we discussed this issue as well. For this years spring special we therefore decided to create a new NGS Favourite – a one stop solution for Amplicon sequencing. By sequencing your amplicons on the GS Junior we will be able to deliver the data in a short turnaround time while you will still profit from the long reads the FLX chemistry provides. Furthermore you will get comprehensive bioinformatic data that will be able to answer already questions like: how many clusters were obtained or what is the homology of each read compared to the representative read. This data will help you for example to analyse metagenomes in your environmental samples like soil, water or gut.

Additionally we as a service provider for oligonucleotide synthesis, gene synthesis, custom DNA sequencing and NGS are able to offer you primers for free for your amplicon sequencing approach if you order our Spring Special.

Contact us if you are interested in a spring special quote or read more on our website. This offer is valid until 30.06.2012.

Many Older Sequencer Are Not Upgraded: Are they Not Used?

Recently Illumina reported a drop in preliminary Q3 revenues. They expect 1% less revenue compared to previous Q3, while Wall Street expected 17% growth (genomeweb). Illumina explains this with uncertainty in funding and higher throughput of the V3 sequencing Kit. They also report that the upgrade rate from Genome Analyzer to HiSeq 2000 was lower as expected and that the use of Genome Analyzer reagents dropped significantly.
In an earlier post I have already speculated that many sequencer are only used at a low percentage of their capacity. This might explain the low upgrade rate of older sequencer and the drop in use of reagents. They are simply not in use.
I have currently no information about the upgrade rate of Roche GS FLX/454 sequencer for use of the new FLX+ chemistry.
Did you upgrade your sequencer already?

GS FLX+ Sequencing Successfully Established

Being the first service provider in Germany having our GS FLX sequencers updated to GS FLX+ technology, we have meanwhile successfully established the sequencing of shotgun, long paired end (LPE) and cDNA libraries with GS FLX+. The FLX+ business is fully integrated in the daily routine of our production team and we like to share our experiences on run performance with you:

Of course the read length differs in dependence on the organism to be sequenced, but average read length of 600-700 bp has been proven to be a realistic value for sequencing of shotgun libraries so far. Also the announced yield of at least 1 million reads per full run could be confirmed in practice: All shotgun runs performed so far surpassed this specification.

When sequencing a shotgun library, data output per segment therefore almost doubled, making the Roche 454 sequencing more attractive again. Sanger-like read length are of very high value for any de novo sequencing approach whether for a genome or a transcriptome project.

When you are interested in our FLX+ sequencing service we invite you have a look at our current special GS FLX+ offers.

Roche Versus Illumina: Where is it Going?

Six and five years after the commercial launch of their first next generation sequencers, the companies Roche and Illumina are still at the forefront of the next generation sequencing market. By today, Roche technology has enabled 1209 peer-reviewed publications while Illumina technology has even enabled 1631 publications (from both companies’ websites).

The current sequencer of both companies are the Roche GS FLX with an average read length of 350-450 bp and 400 Mbp data output per run and the Illumina HiSeq 2000 with read length of up to 100 bp and up to 320 Gbp data output per run.

Both companies’ focus on technological improvements is mainly on enhancing read number and read length. Illumina has recently launched the 150 bp read length (for GAIIx only) and has further announced doubling of yield for Q2 in 2011. Roche is planning to launch the extended average read length of 750 bp sometimes before the end of June according to GenomeWeb News on April 19th, 2011.
In my point of view third generation sequencing will not replace these technologies but enable additional applications as well as combining third generation and second generation technologies for specific project layouts.