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.

What are your intentions when being interested in a bacterial or fungal de novo genome sequencing project?

Typical answers we get from our customers:

• Easy working with the data
• Data suitable for high quality annotation
• Resolution of structural rearrangements
• High consensus accuracy
• High cost-efficiency

All these requirements can be fulfilled perfectly when combining Roche GS FLX++ and Illumina data. The long Roche FLX++ reads of up to 1100 bp give much longer contigs than Illumina reads only do. For scaffolding and to be able to resolve structural rearrangements we sequence shotgun (SG) and LJD libraries with Illumina technology. The adding of Illumina reads keeps the overall costs at a reasonable level. Furthermore the reads correct the Roche sequencing errors at homopolymer sites and therefore enable us to build a consensus sequence with high accuracy.

The superiority of such a hybrid assembly becomes quickly apparent when looking at the following results of one of our proof of concept studies. In this de novo project, we sequenced a fungal genome of about 30 Mbp and approx. 57% GC content. Using the hybrid strategy we obtained only 10 chromosome-sized scaffolds (see figure below) with up to 8.3 Mbp. Remarkably, the 10 scaffolds represent the majority of genetic information present, given that they make up 99.6% of all scaffold sequence information.

Such results enable easy data handling and definitely are an excellent starting point for annotation and studying of gene content and rearrangements.

Sequencing strategy: SG library with FLX++ (approx. 10-fold coverage), SG and LJD 3 kbp, 8 kbp and 20 kbp on Illumina HiSeq 2000 with 2x 100 bp module.

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…

With the update of our MiSeq system to 250 bp reads genome sequencing on this system gets even more important. But long reads and huge data output are not the only prerequisite for a great de novo assembly result.

What is missing?

Paired-end libraries that span gaps and repetitive structures can improve de novo genome assemblies tremendously. Our proprietary long jumping distance libraries (LJDs) are perfectly suited for scaffolding on Illumina sequencing devices. In contrast to other paired-end libraries (like Illumina mate pair library), our LJD library preparation involves an adaptor-guided ligation of the genomic fragments. The different preparation protocol offers the following advantages:

• No hybrid reads – a unique sequence identifies the crossover points
• No shotgun pairs – less than 1% of all LJD reads are shotgun paired-end reads
• Distinct insert sizes – we prepare LJDs with 3, 8, 20 or even 40 kbp insert size
• Span large repeats – large and complex repeats up to 40 kbp can be resolved

Mapped reads: All reads from a 3 kbp LJD library (grey) are aligned to a reference sequence. Two LJD read pairs are highlighted (blue + black) and their measured insert size is 3107 bp and 3002 bp respectively.

Why should I combine MiSeq long reads and LJDs?

The new features of the MiSeq (250 bp reads; data output up to 8 Gbp) enable the combined and cost-efficient approach of shotgun and LJD libraries in one run. The MiSeq output is sufficient to sequence several bacterial genomes or single fungal genomes (up to 60 Mbp) with appropriate coverage.

• Longer reads – more sequence information to correctly map the reads onto your contigs
• Short delivery time – due to the shorter run time compared to the HiSeq 2000

Read more about our long jumping distance libraries on our website

Illumina is currently in the midst of the MiSeq sequencer updates. The software update, the new flowcells and the new sequencing chemistry enable runs with outputs of around 8 Gbp and 250 bp read length. The first updates have reached Europe just recently and only a few days ago our own MiSeq has received the update.

That’s not the end of the story for Illumina. Just a week ago, they already have announced the next update. In the second half of 2013 Illumina is planning to offer another MiSeq update that will increase the output to 15 Gbp. They achieve this tremendous output for their benchtop device by increasing the read length to 300 bp and resolving about 25 million clusters on the flowcell.

Considering the intense competition with Life Tech’s Proton and Ion Torrent sequencer, Illumina needs to steadily improve the specs of their sequencing devices. In March, Life Tech plans to increase the output of their Proton sequencer to around 36 Gbp. That’s still a bit more than the new MiSeq upgrade can deliver, but one also has to evaluate the differences in the read length. While the MiSeq will be able to produce 300 bp reads soon thereafter, the Ion Proton is generating reads from 100 to 150 bp. And the difference is even more remarkably when the sequencing on the MiSeq is performed with the paired-end module – an approach that is not possible with Life Techs devices. By using library insert sizes of around 450 – 500 bp, the two overlapping reads can generate a single consensus read of about that size.

In my opinion the Illumina MiSeq is at the forefront of the race and if Illumina’s plan works out they will be there in 2013, too. But we all know how short-lived the NGS market is. So let’s see what’s coming!

We asked for your opinion if it is possible to directly compare the benchtop sequencers MiSeq and IonTorrent with each other. The result was pretty interesting because the 34 votes were distributed nearly evenly.

Recently, we have reported on the Studies of the Broad Institute, showing that the PacBio RS system was able to outdo MiSeq sequencing regarding validation of SNP analysis. Now Pacific Biosciences have taken another important step to further improve their product.

Pacific Biosciences have now launched a new Sample Loading Device for the PacBio RS, called MagBead Station.  As  Michael Hunkapiller, Ph.D. President and Chief Executive Officer of Pacific Biosciences told in their press release, they expect that with the new device, customers will  “be able to generate 10 kilobase-sized libraries using as little as one microgram of sample, a five to 10-fold improvement from where we were just a few months ago”. Also, because the new process is more robust, they expect that sequencing results will have higher overall consistency, allowing to run experiments also on challenging samples.

First experiences of early-access-customers seem to underline these expections:

As Patrick Hurban of Expression Analysis told InSequence, the new loading device allowed them to recover sequences also for “difficult” samples: “we’re much more confident on a sample-by-sample basis that we will be able to get good sequence”, he said. Also, they could confirm that the amount of library that needs to be loaded is now significantly lower. The new loading process also seems to favor longer DNA fragments over shorter ones, excluding short contaminating DNA fragments. This results in a greater percentage of long reads in a run. Also, the loading process now seems to work as efficiently for the large insert libraries as it does for the smaller insert libraries.

With the new loading device, about 50-60 % of the ZMWs are now active after loading. This is a great improvement compared to 30-45 % of active ZMWs before the upgrade.

When PacBio started on the market, I was impressed by the sophisticated new technology. However, the results of the first projects were rather disappointing. The new loading device now seems to greatly improve the sample loading step. However, the high error rates still remain a challenge, with about 15% for the time being. Pac Bio will need to solve those issues if they want to be successful on the market in the long run. However, it seems that by and by, PacBio is overcoming  its “childhood diseases”.

We are happy to extend our next generation sequencing capabilities with the new Illumina HiSeq 2500 and Illumina MiSeq. Thanks to these new machines, we can provide customers with unsurpassable NGS services at high accuracy rates and shorter turnaround time (TAT). Read more at http://bit.ly/OTv9AP

As already mentioned in our latest blog post Michael Quail and his team from the Sanger Institute published a comparison of the Ion Torrent PGM, the PacBio RS system and the Illumina MiSeq (BMC Genomics). This study and all the others performed recently couldn’t determine one clear winner as each system has its own advantages.

But really interesting are now the statements of the spokespersons from the different companies in a recent article from Julia Karow in GenomeWeb. They all agree on the same thing: the data collected in the publication have been true in 2011, but are outdated by now since a lot of effort is put into innovation. Every instrument performs a lot better now. So what is our conclusion? That comparisons for NGS technologies are just a waste of time? For the Sanger institute it means that they invested in 3 new MiSeq’s since the Illumina pipeline is already available. For me, these comparisons are also valuable for all other institutes. Although maybe outdated, they highlight the strength and weaknesses of each technology and help to decide where to invest thousands of dollars. What do you think?

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.