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Whose Genome Has Been Sequenced? Theobroma cacao L.

Stephanie Engel — Mon, 17 Jun 2013 13:34:08 +0000

I suppose there is no human being on the planet not knowing chocolate. “The tropical Theobroma cacao tree has been cultivated for at least three thousand years. Its earliest documented use is arount 1100 BC (wiki.org).”

The latest de novo genome sequencing publication about a cacoa plant focusses on the Theobroma cacao L. Matina 1-6 clone, which is the most common cultivated type of cacao worldwide (Motamayor et al.). And although a first draft of this clone has already been published in 2010 the authors aim for an improved version of the genome to identify candidate genes regulating traits.

What was sequenced?

Leaves from Theobroma cacao L. Matina 1-6 clone; haploid genome size ~0.5 Gbp

Sequencing strategy: Whole genome sequencing plus BAC & fosmid end sequencing

Libraries: shotgun and 8 long paired-end (LPE) libraries (insert size: 3 kbp; 6 kbp, 8 kbp) on the Roche GS FLX; three fosmid libraries and three independent BAC libraries with Sanger Sequencing
Read output: > 32 million reads
Data output: 711 scaffolds with a total scaffold length of 346 Mbp with a contig N50 length of 84.4 kbp and a scaffold N50 length of 34.4 Mbp
Bioinformatics: Beside other tools Arachne, Megablast and blastx were used for genome assembly

Gene annotation and orthology analysis

Libraries: long normalised libraries sequenced on the Roche GS FLX and short-paired reads libraries sequenced on the Illumina platform
Read output: ~ 7 M reads from the Roche and ~ 1 billion reads from the Illumina sequencing
Bioinformatics: Transcriptome assembly using the NCBI TSA within BioProject 51633 & final refining using PASA. Further tools where used for marker identification and comparison to other plant species

As further analysis tools re-sequencing as well as qPCR expression analysis were performed to finally report a “high-quality sequence and annotation of T. cacao L. and demonstrate its utility in identifying candidate genes regulating traits.” (Motamayor et al.)

From my point of view this is a high complex study using a comprehensive range of sequencing technologies. This shows once more that not only one sequencing strategy is needed to fully characterise a genome and start interpreting its secrets.

Read the complete publication here.

Whose Genome Has Been Sequenced? – Recent posts:

Ion Torrent or Illumina?

Iris Cheung — Tue, 11 Jun 2013 06:49:57 +0000

If you are choosing between Illumina and Ion Torrent for sequencing of your genome of interest, a study published recently by the Broad Institute (Ross et al 2013 Genome Biology 14:R51) may be of interest to you.

The study compares Illumina MiSeq, Ion Torrent PGM and PacBio on sequencing bias in regions with extreme GS content (<10% and >75%) or long AT dinucleotides in three different bacterial genomes. Relative coverage by each technology is lower in all of these difficult regions, but coverage bias was found to be the most pronounced in Ion Torrent PGM data. PacBio demonstrates the least coverage bias, likely because of its amplication-free protocol, but a much higher error rate than the other two platforms was observed. The results are consistent with an earlier study that also compared those same sequencing platforms (Quail et al 2012 BMC Genomics. 13: 341).

Therefore, depending on the characteristics of your genome of interest, your choice of sequencing platform will influence your downstream analyses.

High-Throughput Sequencing Machines By Platform

Carola Grimminger — Tue, 30 Apr 2013 09:34:38 +0000

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!

The British Ash Tree Genome Project

Carola Grimminger — Tue, 23 Apr 2013 09:22:16 +0000

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

800 bp Read Length For Amplicon Sequencing Is Not Science Fiction

Stephanie Engel — Mon, 22 Apr 2013 08:56:05 +0000

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.

Survival Of The Fittest – NGS Library Prep Methods

Stephanie Engel — Tue, 16 Apr 2013 07:27:15 +0000

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.

Synthetic DNA – Data Storage For Eternity?

Georg Gradl — Sun, 24 Mar 2013 15:21:15 +0000

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

Genome Sequencing Analysis of Ash Tree – Supported by £2.4 Million

Carola Grimminger — Thu, 14 Mar 2013 09:25:06 +0000

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 Galaxy of the Genomics Virtual Lab

Carola Grimminger — Thu, 07 Mar 2013 13:21:26 +0000

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:

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

Dear NGS experts, dear blog readers,

Carola Grimminger — Fri, 01 Mar 2013 13:05:04 +0000

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