NGS Expert Blog

About a year ago my colleguage Regina reported about the new possibilities of using the MiSeq system for amplicon sequencing (16S Amplicon Experiments: Which Platform to Choose?). Now, one year later still everything is true about the advantages of amplicon sequencing using the MiSeq (e.g. lower cost/base).

The main advantage of the Roche system are the long reads that are highly valuable for some applications. By ligating appropriate sequencing adaptors we can currently deliver average read length of up to 700 bp when using the GS FLX+ pipeline. Further improvements regarding the read length can be expected with the launch of a new amplicon pipeline from Roche for the Roche GS FLX+ system (planned for summer 2013).

And beside the ultra long reads on the GS FLX+ system there are still some advantages of amplicon sequencing using the GS Junior system compared to other technologies:

+ short turnaround time (starting from 5-10 working days)

+ competitive pricing

+ moderate to long reads (350 – 450 bp)

+ sufficient data output for all projects with a medium size of samples (e.g. up to 24)

What is your preferred next generation sequencing technology for amplicon sequencing? Take part in our current poll.

Newts have an extraordinary ability to regenerate tissues. For example, they can re-grow fully functional limbs after amputation. In addition, regeneration of parts of the central nervous system, the heart, and the lens has been characterized, making them an excellent model organism for studying regenerative processes. However, because of their enormous genome size (10 times that of human), the molecular mechanisms behind this amazing regenerative process are largely unknown.

A research group at the Max Plank Institute recently published a de novo assembly of the transcriptome of the urodelian amphibian Notophthalmus viridescens (Looso M. et al. ). The researchers combined 454, Illumina, and Sanger sequencing data from both normalized and non-normalized cDNA libraries. The resulted transcriptome comprises over 120,000 non-redundant transcripts. Homology search using BLAST led to annotation of 38,000 transcripts. Importantly, they found 800 transcripts, whose protein-coding potential was validated by mass spectrometry, that show no similarity to any know transcripts or show similarity to urodele-specific EST sequences. Some of these transcripts belong to novel protein families.

It is an interesting hypothesis that some of those newt-specific proteins may provide mechanistic insights into regeneration processes unique to these animals. Their work will definitely be an important resource for subsequent studies in tissue regeneration and may benefit future research in regenerative medicine.

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…

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.

It seems that Next Generation Sequencing (NGS) is as seasonal or innovative as fashion. Early this year Oxford Nanopore Technologies announced a revolutionising technology where NGS can be performed on very small sequencers in USB-Stick format.  Just recently Complete Genomics reported about a new technology, named Long Fragment Read (LFR). LFR enables to increase the sequencing accuracy by 10-fold and reduces the amount of starting material at the same time. Additonally QIAGEN disclosed the acquisition of Intelligent Bio-Systems Inc. (IBS). A previously undisclosed NGS benchtop sequencing instrument, combining IBS and QIAGEN technology will be launched soon. The main focus of this technology is the processing of multiple samples in parallel. I am looking forward to learn more about this technology, which seems to have some things in common with the Illumina technology, since Illumina just sued QIAGEN for infringement of NGS patents.

So do we have an excess in development for Next Generation Sequencing or do we need more? My personal opinion is that innovation is the source of science and that it is important to develop new technologies. But it remains fascinating if and when any of the new technologies will replace any of the currently used technologies, like Roche GS FLX or Illumina. I’ll keep you updated.

• 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.

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”.

Just after posting my last update about the fight between Roche and Illumina I read two more interesting news which I would like to share with you.

1. Three important shareholder advisors aid Illumina’s board. Glass Lewis, ISS and Egan-Jones recommend that all shareholders shall support the board by not voting for any of Roche’s candidates to extend the Illumina board. From my point of view this is a clear announcement towards Roche.
2. The current behaviour of Roche is not new. They acquired already two companies in the same way: Genentech & Ventana. Roche always needed to increase the offer per share once and in both cases it took more than 6 months until the deal was signed. Since the discussion about the takeover is public since January I won’t expect a solution now before autumn this year.

On the annual Illumina shareholder meeting this week we will definitely learn how the shareholders react on the recommendations of all advisors and on the offer from Roche.  Will they adopt a clear position? We will know that soon.

The Neandertals lived around 30.000 years ago. The Oetzi died around 5.000 years ago. For both human ancestors researchers were able to fully sequence the genome now. Prof. Pääbo and his group from the MPI in Leipzig published around ~60% of the Neandertal genome in Science (2010). And quite impressively from my point of view is that they give full access to the genome to everyone: they simply put all data on their website. What also fascinated me is that it is quite difficult to study the resemblance between the Neandertal and modern humans since most of the bones found from the Neandertal are “contaminated” with modern human genes. And of course this is obvious since no anthropologist is wearing gloves by default and therefore all people touching the bones to do studies about age and the lifestile of our ancestors will leave their genes on the bones.

An eye opener for me is also that the most obvious thing we discover in the genome is always the impairment of a species. A good example is the recent publication of the complete genome of the Iceman (Oetzi). 96% of the Iceman’s genome has been sequenced and what did we learn: he belonged to blood group O, was lactose intolerant, had probably a genetic tendency towards coronary heart disease, and was carrying Lyme disease.

But researchers also found interesting information about the linkage of both Oetzi and Neandertal to modern humans:

The genome of Oetzi has been compared to 1300 Europeans, 125 North Africans and 20 people from the Arab peninsula. The study revealed that his closest living kin are found on Sardinia and Corsica.

For the Neandertal five modern humans from different populations were used for comparison studies. The stunning result is that some Neandertals and early modern humans interbred since 1 to 4% of the DNA of many humans who live outside of Africa originate from the Neandertal.

In all the discussions about our ancestors and close relatives I sometimes come to think if we will be close relatives in let’s say 1 million years? Wouldn’t it be possible that a new population or species of humans develop? It sounds absurd or science fiction-like but who are we that we think there is nothing “after us”?

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?