NGS Expert Blog

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

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.

Whole Genome Mapping (WGM) using the OpGen Argus technology delivers high resolution, ordered whole genome restriction maps from single DNA molecules. To receive such a restriction map it is crucial to isolate long DNA fragments (200 kb in size and longer) and to capture the DNA on a solid phase. Afterwards the DNA is digested revealing restriction cleavage sites as gaps when using a fluorescence microscope to visualize the DNA. This optical map will then be converted into digital data, the so called “single molecule restriction maps” (see video below). The software MapSolver enables the following analysis options (see details in the analysis video):

• Perform Genome Comparisons
• Identify Motifs, Annotate Features, and view in silico sequence data
• Perform Sequence Placement
• Create Similarity Clusters

 Video about step 3: How to scan and assemble single molecule restriction maps (SMRM)

Recently we gained access to this innovative technology and are able to combine our Next Generation Sequencing Service with the WGM technology. The combination of NGS and WGM can be used to order the contigs from a next generation sequencing project against the optical map scaffold. This method is able to highly improve sequencing assemblies. If you are interested in a combined or stand-alone project for WGM, please do not hesitate to contact us.

We look forward to discuss WGM in detail with you.

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.

After having launched the new C2 chemistry for PacBio RS sequencing with longer read length it has been quiet for a while with Pacific BioSciences. However, a few days ago they have again attracted attention by launching a new analysis software that indicates base-modifications in the sequencing data. And from what I hear and read about these techniques, the epigenetics market could really be a great success story for Pacific Biosciences.

As PacBio’s SMRT sequencing is observing the DNA polymerization in real time it allows not only to decode the sequence, but also to study kinetic characteristics of the process. The kinetics of a base incorporation is characteristically changed by the presence of a modified base in the template strand and therefore can be used to distinguish between different base modifications. Different modifications result in different signatures (or fingerprints) that vary in signal magnitude and the length of the region over which the kinetics are altered.

I think that the study of base-modifications with PacBio RS has several advantages compared to experiments like methyl-Seq or bisulfite sequencing. On the one hand side PacBio RS sequencing is a direct detection, where no enzymatic restriction or bisulfite conversion has to be applied upfront. On the other hand – and this is the most important advantage for me – the PacBio RS system allows to distinguish a wide spectrum of base modifications, which has not been possible so far.

Unfortunately, the recently launched software is not yet ready to distinguish the different types of modifications, it only flags positions where modifications are present. However the company has shown proof of principle data and has already stated that the information to discriminate between modifications will be incorporated into future releases of the software. Moreover a Technical Note is provided from the company regarding their motif identification tool for bacterial methylomes.

Rather than resting on the successes of the MiSeq launch, Illumina is continuously improving the performance of their small Benchtop Next Generation Sequencing System. Geoff Smith is talking about improvements in the read lenght of the MiSeq in the video attached.

This instrument is not only another next gen sequencing device but really has remarkable advantages over other instruments when sequencing for example bacterial genomes. This is why I am really delighted that we now can offer services using the MiSeq instrument. (More info on our MiSeq services can be found here)

Last week The Smithsonian Institution’s National Museum of Natural History at Washington DC announced a new exhibit to celebrate the 10th anniversary of the completion of the human genome. The project is a collaboration between the museum and the National Human Genome Research Institute, with major funding coming from the Life Technologies Foundation. It will open in 2013 to the 7 million annual visitors of the museum.

“The goal of the exhibition is not just to celebrate but to look ahead and acknowledge that we are in the early stages of a very exciting genomic era, that we have learned a remarkable amount about how the genome works and how it contributes to health and disease, and that the pace of research is only accelerating and becoming increasingly relevant to people,” said NHGRI Director Eric Green.
Also announced last week was a new grant program by the NHGRI to study newborn genome sequencing. It will provide $25 million to study how whole genome or whole exome sequencing will benefit newborn care as well as its social implications.
“Genome”, “genomics” etc used to be terms understood by few outside of biology and bioinformatics. This is changing rapidly. It is exciting time ahead of us to witness the genomics revolution.

My last week’s blog article was about expression profiling with mRNA-Seq libraries and about the required sequencing depth of this protocol. But there are other possibilities for expression profiling, and today I especially want to highlight the 3’-fragment library protocol.

The big advantage of this protocol is that it provides a much higher resolution than mRNA-Seq does. The reason is that within mRNA-Seq the average transcript is represented by approx. 10-25 reads that cover the whole transcript, while with the 3’-fragment protocol only one read is generated per transcript. The derived reads from a 3’-end library map to the 3’-end of the transcripts and expression differences are easily collected by just counting the reads that map to a specific reference transcript.

The 10-25-fold higher resolution comes along with considerably reduced projects costs as 10-25-fold less sequencing is required to obtain a similar depth of the analysis. Or in other words: When analyzing the same number of samples per channel the 10-25 fold higher resolution allows the scientist to even look at very low expressed genes with reliable statistical evidence.

Of course the mRNA-Seq protocol is needed in case other analysis shall follow, like the study of alternative transcripts, or fusion genes. But this is anyway a completely different story as these applications need an even higher sequencing depth than expression profiling with mRNA-Seq does require.

As a conclusion I think it is definitely worth to evaluate this protocol when having in mind an expression profiling experiment. And we would be delighted if you share your thoughts on this with us and the other blog readers.

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.