NGS Expert Blog

While data output and quality of Next Generation Sequencing is continually increasing, the cost per base is steadily dropping. A survey  from the National Human Genome Research Institute (NHGRI) shows that the cost development even exceeds Moore’s law. New doorways  for research are opening, which may not have been regarded as realistic in the past  due to this trend.

For example, over the past years, several approaches have been made to use DNA as a means of storing information. In a study recently published online in Science, scientists developed a strategy to encode and read digital information using DNA Synthesis and  Next Generation Sequencing Systems.

A html document containing more than 50,000 words, 11 JPG images, and a Java Script program was encoded in DNA by synthesizing nearly 55,000 oligonucleotides on high-fidelity microarrays. The information stored in the oligonucleotides library was later “read” by Illumina sequencing.

According to the authors, DNA is a very useful medium for long term storage of information:   DNA is very stable over many years,  allows data storage at very high density and  small volumes. The senior author, Kosuri, told InSequence, they only used some 50 ng of oligonucleotides to store the information of this html document! Kosuri admitted that the study costed several thousand dollars. However, if Next Generation Sequencing continues to develop at the same speed as today, new applications such as using DNA for (long-term) data storage may become a feasible option.

So let us see what is coming next!

Next-generation sequencing allows detection of minor variants in a heterogeneous sample. However, errors in PCR and sequencing pose limits on its sensitivity.
A group at University of Washington developed a method, called Duplex Sequencing, to dramatically improve accuracy by sequencing both strands of each DNA duplex. Mutations that are detected in the consensus sequence of one strand but not the other are discounted as technical errors.

The authors adopted the method to Illumina sequencing. It involves the use of modified adaptors that have a tag with random sequence attached. After ligation of these modified adaptors, each duplex DNA fragment is flanked by two different tags and subjected to paired-end sequencing. Sequences of the same duplex from the complementary strands can therefore be uniquely identified by having the same tags on either ends. Comparing sequences of the two strands allows identification of true mutations. The authors estimated that Duplex sequencing has a theoretical background error rate of less than one per 10⁹ nucleotides sequenced.
Full text article can be accessed here: http://www.pnas.org/content/early/2012/07/31/1208715109.full.pdf

Dr. Georg Weinstock from the Genome Institute at the Washington University presents in a webinar how to create the perfect genome assembly by using the optical mapping system from OpGen Inc.

A recent article in Scientific American by Jennifer Ackerman entitled “The Ultimate Social Network”, highlights a particular problem when trying to sequence the genomes of eukaryotic organisms. The problem is that the organism in question, whether it is an ant, butterfly, a polar bear, frog or Blue whale is not a singular organism at all.

In fact the organism in question plays host to many millions of other prokaryotic organisms, mainly bacteria, viruses, fungi or parasites. In humans for example the genetic material from the microbiome outnumbers the human genome by at least 10 to 1. This is also expected to be true of all other eukaryotic species which harbour and maintain a symbiotic relationship with their microbiome.

The genes from the microbiome help process beneficial compounds and act to temper host immune defences for example. Therefore, when taking and extracting DNA from a eukaryotic organism it has to be considered what other genomes you may be preparing and sequencing alongside the desired genome of interest. For example it cannot be simply a case of freeze drying an insect crushing into a powder then extracting the DNA, as the resultant samples will contain a highly mixed and diverse set of genomes,  whereby the genome of interest may be present only in the lowest possible ratio. So, be warned! When assembling genomes be sure you know what your starting material actually contains.

Next Generation Sequencing (NGS) is transforming today’s genomic research and is used in numerous applied areas from clinical diagnostics to academic research. In Texas USA, Dr. Steven Phelps and his research team recently used NGS sequencing to discover a gene which allows mice to communicate by singing a song. I have to admit it sounds more like screaming than singing to me. But Phelps and his team found out that a gene called FOXP2 is responsible for this way of communication.

Phelps’ uses next-generation sequencing to decipher how FOXP2 interacts with DNA to regulate the function of other genes. The process involves reading tiny fragments of overlapping DNA so that the entire sequence can be deduced. It is a procedure that generates massive amount of data that only the processing power of a supercomputer can handle, said O’Connell (Source: www.tacc.utexas.edu). So data handling & storage is still one of the biggest challenges when performing Next Generation Sequencing projects. But now take the chance an listen to the song of this little mouse.

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.

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.

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.

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”.