NGS Expert Blog

The High-Throughput Sequencing map by James Hadfield (Cancer Research UK, Cambridge) gives us a very interesting overview about sequencing activities around the world. We ran a survey to find out if your favourite machines correspond with the platforms listed by James in his overview.

Here are the results: Your personal favourites are nearly a perfect match with platforms in the genome centers worldwide. Great match!

Mid of March we wrote about the British Ash Tree Genome Project.

Yesterday, the School of Biological and Chemical Sciences, Queen Mary University of London launched a website explaining in detail their amazing project: Find there general information, data and tools. Further, an interview on the project from the Radio 4 Today programme on 21/12/12 can be heard here. More details on the project can be heard on NERC’s 5/2/13 Planet Earth podcast.

Visit http://ashgenome.org

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.

30 years of PCR in various applications has revolutionised molecular biology. But PCR also has its drawbacks. One of them is the amplification of AT- or GC-rich DNA fragments. Naturally, researchers are often interested in sequencing and studying genomes with high GC or high AT content, like S. aureus with a AT content of 67% or Streptomyces coelicolor with a GC content of 72%.
But more and more NGS kit providers try to circumvent PCR in the library prep. Ashley Yeager has summarised the current status of PCR-free library preps including a comprehensive overview of the pro’s and con’s of both methods (BioTechniques).

Summarising the findings from Mrs. Yeager there is no clear champion in sight:

Library prep by using PCR methods
+ well-known lab procedure & good sequencing efficiency
- difficulties in amplifying GC- / AT-rich regions -> sequencing is biased

PCR-free library prep
+ good sequence read distribution & a more even genome coverage
- huge amounts of starting material needed & sequencing reaction is less efficient

Read the complete article under BioTechniques.

In the April issue of the journal Spektrum der Wissenschaft I found a very interesting article from Jan Dönges about data storage of information with the help of synthetic DNA (oligonucleotides). He describes the work of Ewan Birney and Nick Goldman from the European Bioinformatics Institute (EBI) in Hinxton, UK who have developed a strategy that allows coding data in strings of A, C, G and T nucleotides (Nature 494, 77-80, February 7 2013). They coded all sonnets of Shakespeare, a photo of the institute, the original paper of Watson and crick about the structure of DNA, an audio recording of the speech of Martin Luther King “I have a dream” and file with coding instructions; all together 739 kilobyte of information. They ordered the oligos and sequenced them on an Illumina HiSeq 2000. They received a text file of the letters A, C, G and T that could be converted into the original data. The complete code and sequence can be found here.

From sequencing experiments like the mammoth or the Neanderthal man we know that DNA is at least 10,000 years stable, longer than any other data storage. In addition it is extremely dense. With 1 gram of DNA it is possible to code more than 2 petabyte (10¹⁵ byte), or 2.3 million gigabyte. The volume of a coffee cup would be sufficient to code 100 million hours of high resolution videos. It is to be expected that the technology could even be improved in the future as long as mankind still is interested in DNA. The cost for the experiment was quite high compared to other storage media like tapes, HDD or DVDs. However, already after 600 years of making consecutive security copies of tapes the cost is compensated. So, if we want to conserve the knowledge of mankind for very long periods and make sure that it survives possible major disasters in the future, this seems to be a reasonable strategy

To conduct genome sequencing and analysis of Ash (Fraxinus excelsior), researchers in the UK received £2.4 million ($3.6 million / €2.8 million). The major aim of this project is to increase the understanding of the wide spreading fungal tree disease, which is widespread in northern Europe and has already been found at more than 300 sites across the UK (see http://www.forestry.gov.uk/chalara). Those fungi attack ash tress but some tress resists those attacks.

For this reason a lot of samples of the ash dieback fungus will be sequenced and – funded by an urgency grant from the Natural Environment Research Council – the complete genome sequence of Ash is aimed to be available by August.

Sequencing of the approximately 900 Mb plant genome will be performed applying the latest hybrid de novo sequencing strategy, recently proven to deliver excellent scaffolding and assembly results. This new golden standard in de novo sequencing employs a combination of Roche/454 FLX++ long read technology (software version 2.8 with read lengths up to 1,100 bp) and Illumina HiSeq 2000/2500 high throughput sequencing with several ultra-accurate long jumping distance libraries (LJD of 3kb, 8kb, 20kb and 40kb), supplemented by sequencing of Illumina shotgun libraries with different fragment sizes.

With the sequenced ash tree genome the researchers hope to hold clues to how some of the trees (2% are able to defend the disease) are able to resist attack, and knowledge about the genetic differences between resistant and non-resistant trees. This knowledge could be used to develop trees that can’t be infected.

Project leader, Dr. Richard Buggs from Queen Mary’s School of Biological and Chemical Sciences: “Sequencing the ash genome is a foundational step towards discovering the genetic basis of resistance to ash dieback – the future of ash trees in Britain may depend on this”.

Read more about that exciting project at GenomeWeb about the general project and at Eurofins MWG Operon about the genome sequencing.

The Genomics Virtual Lab (GVL) project – using the computing resources from the NeCTAR Research Cloud – is an Australian Government project conducted as part of the “Super Science” initiative. It is developing infrastructure supporting genome informatics research.

Their Galaxy-based NGS and HTS tutorials are really excellent:

• RNA-seq tutorial
• Variant detection tutorial
• Variant detection (advanced) tutorial
• Genome assembly tutorial

You will love the precise explanations, the hands-on demonstration and the additional material like screenshots and in-depth information!

the last weeks you were asked for your opinion about the NGS Expert blog. We really were overwhelmed when we have read your comments and positive feedback.
Thank you so much for your interest in reading our blog posts.
We absolutely take your feedback to heart and try to focus on your favourite topics. You are the reason why we keep on reasearching to write exciting blog posts!

Best regards
your NGS Expert team

Newts have an extraordinary ability to regenerate tissues. For example, they can re-grow fully functional limbs after amputation. In addition, regeneration of parts of the central nervous system, the heart, and the lens has been characterized, making them an excellent model organism for studying regenerative processes. However, because of their enormous genome size (10 times that of human), the molecular mechanisms behind this amazing regenerative process are largely unknown.

A research group at the Max Plank Institute recently published a de novo assembly of the transcriptome of the urodelian amphibian Notophthalmus viridescens (Looso M. et al. ). The researchers combined 454, Illumina, and Sanger sequencing data from both normalized and non-normalized cDNA libraries. The resulted transcriptome comprises over 120,000 non-redundant transcripts. Homology search using BLAST led to annotation of 38,000 transcripts. Importantly, they found 800 transcripts, whose protein-coding potential was validated by mass spectrometry, that show no similarity to any know transcripts or show similarity to urodele-specific EST sequences. Some of these transcripts belong to novel protein families.

It is an interesting hypothesis that some of those newt-specific proteins may provide mechanistic insights into regeneration processes unique to these animals. Their work will definitely be an important resource for subsequent studies in tissue regeneration and may benefit future research in regenerative medicine.

Discover 2270 high-throughput sequencing machines in 830 centres at http://www.omicsmaps.com/. Select according to a specific platform or search for a facility or a region of your interest. Relevant statistics are also available at http://www.omicsmaps.com/stats.

No matter if you are a commercial service provider, a researcher or a device manufacturer, you will love the map!