NGS Expert Blog

Dear all,

Here is my second summary from 4th NGS Congress at London Heathrow end of 2012. It will bring to you some (hopefully) interesting new facts about sequencing with PacBio RS - the second long read technology present in the actual markets and also the only system delivering reads even longer than 10,000 bp…

Kevin Corcoran, Senior Vice President at Pacific Biosciences held an interesting and very nice talk about the most recent developments for the PacBio RS system. He also showed some nice detailed road maps about future aims and plans. One important thing actual to be mentioned is the launch of the new “XL Chemistry” – while still “C2 Chemistry” may be used as well. The other very interesting story is about “Stage Start” a new feature enabling a parallel start of all sequencing detection similar to the well-known “hot start” technology for PCR. Such detection of sequences better will start from a defined position for most of the libraries than starting from somewhere in the middle. Last but not least, I’m very keen to learn how the future “Photo Protected DNA Polymerases” may further develop – an idea being really very, very next-next-generation…

First of all I can summarize that applying “XL Chemistry” looks really interesting and this being true also in terms of Eurofins MWG Operon de novo sequencing and assembly focus.  This new feature of the PacBio RS machine may also open some new doors to other types of applications, while in general the need for extrem high data coverages may be reduced in parallel.

Currently “C2 Chemistry” is on the machine and running a 90 min video may deliver you about 20-50,000 reads and data outputs of 30-50 Mb – of course higher yields may be possible for “ideal” DNA samples. The average read length is about 3,000 bp (!), while the 95% percentile is about 8,000bp. With the new ”XL Chemistry” we got an average yield of about 40,000 reads per SMRT cell with an average read length of about 4,000bp (+30%). Overall, we are very pleased with these first results, especially since we see some good potential to further increase data yields using the new software pipeline started in parallel (Hierarchical Genome Assembly Process and Quiver).

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It is also important to mention two different ways of “How-to-deal” with the XL Chemistry. 1) ”XL chemistry for Polymerase binding”, but “C2 chemistry for sequencing”. This allows for longer reads at the same quality (currently we still do have a single error rate of 10% to 20 %, average maybe 15%). 2) “XL chemistry for Polymerase binding” AND ”XL chemistry for sequencing”. Such one can yield even longer reads, but unfortunately the error rate will also increase by a few %. Therefore this method is being recommended especially for de novo assembly or finishing genomes.

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Finally one real “next-next-gen” highlight was the presentation of a development at Pacific Biosciences scoping with the idea to protect the polymerase enzyme from being killed by the energy of the laser. A picture shows how this should work in principle - by setting in place a laser-light protecting sun-blocker - this story was really fascinating for me and I hope to see in future more than the very promising first data results …

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So over all Pacific BioSciences keeps also moving very fast in year 2013 and it will be very nice to see and learn how all these additional improvements and new features may  improve the overall data results of this fascinating very long read technology offering today real single reads longer than 10,000 bp.

Cheers now and see you on our next BLOG,
Axel

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…

Dear honourable reader of our NGS blog,

Actually IWF and European country leaders discuss a raise of the European secure funds to about 1.5 Trillion Euro (German = 1,5 Billiarden Euro). This is such a high number that I could not even imagine it…

This number also gave raise to the question: “How much of NGS sequencing or Whole Human Genome Sequencing (WGS) could be done with it?”

Step 1:  How many people are needed to pay 1.5 Trillion Euro of income tax?

With an average of 10,000 Euro of income tax per year 4,500,000 people would need to pay for 35 years (life time of work) to account for 1.575 Trillion Euro (1,575,000,000,000 Euro). Or more than 150 million people (150,000,000) are needed to work and pay tax money for one year – of course without any interest.

The 27 European Union countries (EU27) currently have a population of about 501 million people including babies, pensioners and all other non-tax payers (year 2011). Such about 30% of all people in EU27 need to work for one year to earn this sum of funding – of course without spending money for anything else…

Step 2: How many Human genomes would I get for this sum?

At a reasonable cost of 15,000 Euro per genome this equals 100 million (100.000.000) sequenced genomes  – that is about 1/5 of all EU27 people. At a discounted offer of 5,000 Euro per genome this equals 300 million (300,000,000) sequenced genomes – that is 2/3 of all EU 27 people. At a best price offer of about 4,000 $ (= 3,000 Euro) the money would allow to sequence about 500 million (500,000,000) genomes which is the complete population of EU 27.

Whow – this gives me the feeling we are in fact talking about a lot of money,

Best regards

Axel

NuGen has launched a new kit > to multiplex 384 samples on Illumina HiSeq 2000, while we by today offer on routine  max 96 samples per channel. I guess this is quite interesting new update.

Has anyone  by today already got experience with this new kit? Have you observed any problems during handling? How would you rate this kit?

Cheers, Axel