NGS Expert Blog

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!

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.

Illumina product experts discuss within 4 videos

1. what it takes to get a good genome out of a sequencing system,
2. what attributes of a sequencing system contribute to the quality of a sequencing data set,
3. the hidden costs of accuracy and
4. how next generation sequencing is moving into the clinic because of a better understanding of accuracy.

Please be tolerant, they’re from Marketing

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.

Eric Green is talking about the genomic landscape. Please enjoy the great presentation – starting at min 6.00.

The upgrade of the Illumina MiSeq promises 250 bp reads with a turnaround time in the paired-end module of ~ 35 hours. This upgrade is planned for Q3 so any day now it could happen.

Anyway I think that already the current version is an excellent alternative to other technologies when sequencing of bacterial genomes is of interest.

Since we only have the MiSeq since several weeks we want to add a bonus to bacterial sequencing on the MiSeq in our new special. The special delivers 2x 150 bp paired-end module reads for 2, 3, or 4 bacterial samples. The bioinformatic analysis like mapping against a reference genome and SNP/InDel detection can be ordered optinally. If you are interested in this special order before September 30th and profit from a 10% discount. Read more about our special here.

The Genomatix Genome Analyzer has been chosen by a panel of 30 experts to be awarded.

Genomatix has been awarded the “INDUSTRIEPREIS 2012″ in the biotechnology category at the Hannover Messe (Germany), the world’s biggest technology trade show. They have received it for its data analysis and interpretation platform Genomatix Genome Analyzer (GGA).

“…It’s a big motivation for the whole team to come up with even more innovative ideas on how to deal with the increasing deluge of Next Generation Sequencing data”, adds Dr. Martin Seifert, CEO of Genomatix, “getting an award that explicitly rewards products of technological and social value underlines that NGS is quickly moving from a research tool into the broader field of clinical application.”

Interested in expression profiling, but you are working with a non-model organism?

A very elegant way for this purpose is to (1) generate long cDNA contigs with NGS technologies that serve as a reference transcriptome and (2)  perform expression profiling by mapping Illumina HiSeq 2000 derived short reads of each sample back onto the reference. As only one read is generated per transcript, down and up regulated genes easily can be identified by counting the sequence hits.

This approach was used by Mutasa-Göttgens et al., 2012  in order to analyze targets involved in bolting and flowering in sugar beet. Understanding the regulation of the vernalization-induced bolting and the change towards the reproductive phase is of high importance because bolting and flowering cause considerably reduced sugar content.

To generate the reference transcriptome of the shoot apex, a normalised random primed cDNA library was prepared and sequenced on Illumina HiSeq 2000 with single read module and 100 bp read length. De novo assembly yielded at total of 225’000 unique transcripts, 53’000 of which represent large transcripts (>500 bp and up to >8’700 bp). For quantitative comparison we prepared for the research group a digital gene expression (DGE) library from samples which were subjected to vernalization and / or phytohormone treatment. The libraries were sequenced on Illumina HiSeq 2000 and reads were mapped onto the transcriptome reference sequence.

Bioinformatics analysis identified (amongst others) a potential regulator of vernalization, and therefore an interesting breeding target for the sugar beet crop.

In my opinion, this study is an excellent example of how to combine the strength of different available RNA-Seq libraries most effectively. The normalized random primed library allows unbiased site-directed sequencing. Furthermore the normalization process levels high and low expressed transcripts, which allow identification of low expressed genes accurately and facilitate de novo assembly with short read technology considerably. The DGE library in contrast produces only one tag per transcript, thus allowing much deeper resolution than the mRNA-Seq approach from Illumina, which generates reads that cover the whole transcript.

In the meanwhile, with new NGS libraries available, one would rather use a 3’-fragment library instead of the DGE library. While displaying similar costs, this library type offers longer sequence information (100 bp versus 17bp) and in consequence higher mapping accuracies and reduced numbers of non-mappable reads.

You will find more information regarding this combined approach including the 3’-fragment library for read counting in the following Application Note.