Sunday, 30 September 2012

NGS PubMed Highlights: Puerto Rican Parrot Genome Sequenced

Thanks to a fundraising campaign (small donations from local community), researchers were able to sequence the genome of the critically endangered Puerto Rican Parrot. The study was published Friday in BioMed Central and BGI’s open access journal GigaScience.
Interestingly the Project also has a Facebook page that I guess was instrumental for the fundraising campaign.
I'm wondering how is the sequence of the FOXP2 Parrot gene (just kidding... or maybe not).

A locally funded Puerto Rican parrot (Amazona vittata) genome sequencing project increases avian data and advances young researcher education

Taras K Oleksyk, Jean-Francois Pombert, Daniel Siu, Anyimilehidi Mazo-Vargas, Brian Ramos, Wilfried Guiblet, Yashira Afanador, Christina T Ruiz-Rodriguez, Michael L Nickerson, David M Logue, Michael Dean, Luis Figueroa, Ricardo Valentin and Juan-Carlos Martinez-Cruzado

GigaScience 2012, 1:14 doi:10.1186/2047-217X-1-14
Published: 28 September 2012

Abstract Background
Amazona vittata is a critically endangered Puerto Rican endemic bird, the only surviving native parrot species in the United States territory, and the first parrot in the large Neotropical genus Amazona, to be studied on a genomic scale.
In a unique community-based funded project, DNA from an A. vittata female was sequenced using a HiSeq Illumina platform, resulting in a total of ~42.5 billion nucleotide bases. This provided approximately 26.89x average coverage depth at the completion of this funding phase. Filtering followed by assembly resulted in 259,423 contigs (N50 = 6,983 bp, longest = 75,003 bp), which was further scaffolded into 148,255 fragments (N50 = 19,470, longest = 206,462 bp). This provided ~76% coverage of the genome based on an estimated size of 1.58 Gb. The assembled scaffolds allowed basic genomic annotation and comparative analyses with other available avian whole-genome sequences.
The current data represents the first genomic information from and work carried out with a unique source of funding. This analysis further provides a means for directed training of young researchers in genetic and bioinformatics analyses and will facilitate progress towards a full assembly and annotation of the Puerto Rican parrot genome. It also adds extensive genomic data to a new branch of the avian tree, making it useful for comparative analyses with other avian species. Ultimately, the knowledge acquired from these data will contribute to an improved understanding of the overall population health of this species and aid in ongoing and future conservation efforts.

Friday, 28 September 2012

Flash Report: Interesting improvements in Ion performance

Here is another interesting video from Ion World 2012. Chad Nusbaum from Broad Institute presents their data showing improvements in dealing with indels and homopolymers using the new version of the Torrent Suite software. Even better results are being obtained with the new Ion Proton platform.

Wednesday, 26 September 2012

Exciting news on Ion Semiconductor Sequencing Technology

Take a look to this video from the Ion World 2012 Conference published on the Ion Torrent Channel on YouTube. This is a report of the work done at the Cancer Genome Research of the National Cancer Institute using 6 Ion Torrents and recently also 4 Ion Protons.
It's really exciting to see such a rapid increase in performance and accuracy for the Ion Torrent platform. Moreover high multiplexing protocols based on AmpliSeq panels seem to cut cost per sample to a very cheap price!

These data are even more interesting considering that they are provided from an independent and authoritative source external to Life Technologies.

Tuesday, 25 September 2012

Flash Report: Exclusive deals and discounts: Mexican Dinner for Two, Haircut & Highlights, or ... a DNA test?

It is quite shocking to realize that DNA tests are now commercialized using the same strategies used to sell a Fat-Burning Lemonade Package, Woodies Bamboo Wayfarer Sunglasses or a Three-Night Stay at a Resort Hotel in a nice location.
I just found this deal on the Italian Groupon site for a DNA test for cellulite (€99, approx. $125). Apparently 19 customers already decided to give it a try. Moved by curiosity I found the web site of the MD offering the test.  It is interesting to note that he is also offering, among the others, a Relax DNA test (also called Anti Stress) and a Longevity DNA test. The physician is also President of the Italian Medical Anti-Aging Association. In what appears to be the site of the association, they offer a number of DNA tests (including the ones mentioned above) and they provide a sort of Primer on Genomics stating that "In tutto il genoma abbiamo all'incirca 180.000 geni di lunghezza variabile e molte sequenze di DNA non codificanti" ("in our genome we have approximately 180.000 genes of variable length and several non coding DNA sequences"). The number of genes is even highlighted in bold :-(
I guess I do not need to make any comment.

Monday, 24 September 2012

NGS PubMed Highlights: Oyster genome sequenced!

The oyster genome has just been published on Nature. Besides adding new information on the evolution of the molluscs and providing useful genetic background for selection and aquaculture, this study also used a peculiar sequencing protocol based on fosmid-pooling  and a self-developed assembly strategy to reconstruct the complete genome (instead of the classical short-read libraries plus long-read ones for scaffold assembly).
The oyster genome revealed enrichment in stress-related genes that make this mollusk resistant to environmental stress, and shed light on the complex biological processes implied in the shell formation.

The peculiar sequencing and assembly strategy adopted in the paper

Stress response pathway enriched in the oyster genome

The oyster genome reveals stress adaptation and complexity of shell formation. 
Nature. 2012 Sep 19

Zhang G, Fang X, Guo X, Li L, Luo R, Xu F, Yang P, Zhang L, Wang X, Qi H, Xiong Z, Que H, Xie Y, Holland PW, Paps J, Zhu Y, Wu F, Chen Y, Wang J, Peng C, Meng J, Yang L, Liu J, Wen B, Zhang N, Huang Z, Zhu Q, Feng Y, Mount A, Hedgecock D, Xu Z, Liu Y, Domazet-LoŇ°o T, Du Y, Sun X, Zhang S, Liu B, Cheng P, Jiang X, Li J, Fan D, Wang W, Fu W, Wang T, Wang B, Zhang J, Peng Z, Li Y, Li N, Wang J, Chen M, He Y, Tan F, Song X, Zheng Q, Huang R, Yang H, Du X, Chen L, Yang M, Gaffney PM, Wang S, Luo L, She Z, Ming Y, Huang W, Zhang S, Huang B, Zhang Y, Qu T, Ni P, Miao G, Wang J, Wang Q, Steinberg CE, Wang H, Li N, Qian L, Zhang G, Li Y, Yang H, Liu X, Wang J, Yin Y, Wang J.

The Pacific oyster Crassostrea gigas belongs to one of the most species-rich but genomically poorly explored phyla, the Mollusca. Here we report the sequencing and assembly of the oyster genome using short reads and a fosmid-pooling strategy, along with transcriptomes of development and stress response and the proteome of the shell. The oyster genome is highly polymorphic and rich in repetitive sequences, with some transposable elements still actively shaping variation. Transcriptome studies reveal an extensive set of genes responding to environmental stress. The expansion of genes coding for heat shock protein 70 and inhibitors of apoptosis is probably central to the oyster's adaptation to sessile life in the highly stressful intertidal zone. Our analyses also show that shell formation in molluscs is more complex than currently understood and involves extensive participation of cells and their exosomes. The oyster genome sequence fills a void in our understanding of the Lophotrochozoa.

Tuesday, 18 September 2012

Flash Report: Exome sequencing using the Ion Proton™ System

In a recent Product Bulletin Life Technologies presents some preliminary Exome Sequencing data generated using the novel Ion Proton platform and the Ion PI™ Chip.
The table below summarizes the results obtained.

Interestingly, at the recent at World 2012 customer conference (13 Sept) Rothberg announced that "Life Technologies has developed Avalanche™, a revolutionary 30-minute emulsion-free template preparation chemistry that will work on all Ion platforms. Dr. Rothberg also announced that Avalanche™ will enable an Ion PIII™ Chip for the Proton, with more than 1.2 billion sensors that will take researchers beyond the $1,000 genome".

Flash Report: The Giants' Wedding

Yesterday BGI agreed to acquire Complete Genomics for about $120 million to expand in the U.S. market for medical and research DNA testing.
Complete Genomics’s sequencing technology gives results slower than that of Illumina, however comparative studies have shown that the technology BGI is acquiring is more accurate than Illumina’s. The purchase may also reduce BGI’s dependence on sequencer-maker Illumina.
More details about the takeover at Bloomberg Businessweek.

Monday, 17 September 2012

Rapid development of clinical and diagnostic applications for NGS benchtop sequencers

Besides research projects, there is growing interest in applying NGS technologies to diagnosis and clinical pratice. The new benchtop sequencers have dramatically reduced the costs, making also the analysis of a reduced number of samples cost effective and allowing small center to jump in the NGS field. While the big instruments (like HiSeq 2000 and 2500 or the recently released Ion Proton) remain intended for large sequencing centers or whole genome/exome complex analysis, the small ones (MiSeq and Ion Torrent) are being shifted markedly toward dignostic applications. The final aim for both Illumina and Life Tech is to obtain clinical certification for target resequecing protocols on their machine, even if this goal still seems not at hand in the short period. Indeed, the error and false positive rates of NGS platforms, togheter with lack of standards in the analysis pipeline, remain major concerns and prevent NGS technology from a rapid access to the clinical field.

However, the use of target resequencing and WES has demonstrated to be a powerful tool in molecular diagnosis and mutation discovery. Several papers have been published reporting the identification of new mutations for known syndromes as well as for new rare diseases. Some interesting examples are: - - the use of WES to identify molecular defects in Usher syndrome; (SOLiD, GAIIx and 454 GS FLX)
- the application of 454 technology for the dignosis of inherited retinal degeneration;
- a novel mutation identified in CUL7 gene in a patient with an undiagnosed growth disorder (HiSeq 2000);
- application of WES in a consaguineous family that lead to the identification of mutations in GTDC2 gene as cause of Walker-Warburg Syndrome (HiSeq 2000);
- identification of KCNQ2 defects as cause of Ohtahra syndrome, a kind of neonatal epileptic encephalopathy (HiSeq 2000);
- identification of the molecular defect in the calcium channel gene CACNA1S in a children with atypical episodic muscle weakness (HiSeq 2000);
- discover of compound heterozygous mutations in the PKHD1 gene as cause of recessive polycystic kydney disease (HiSeq 2000);
- identification of missense mutations in SLC29A3 gene as cause of Dysisteosclerosis (HiSeq 2000), a peculiar form of ostepetrosis;
- study on a large family affected by essential tremor neudegenerative syndrome that revealed defects in the FUS gene (SOLiD);
 - discovery of ANK3 mutations as associated with autism syndrome applying WES to a cohort of about 60 individuals (GAIIx).
These publications indicate that Illumina HiSeq sequencers dominate the NGS arena by now. We are curious to see if something is going to change with the capillary installation of Ion Proton sequencers.

A case worth mentioning is for sure cystic fibrosis, a disease caused by impairment of the CFTR gene with more than 1000 mutations identified so far. Even if a major percentage of cases are due to a restricted set of about 20 mutations, the possibility to quickly sequence the entire gene would simplify and speed-up the molecular diagnosis, particularly for ethnic groups with a high prevalence of rare mutations. In the past year some paper came out describing the application of Ion Torrent PGM sequencer for a quick and rapid detection of CFTR mutations and the set-up of a diagnostic protocol based on this NGS platform (see a recent example here). Recently, the University of Medicine and Dentistry of New Jersey's, Institute for Genomic Medicine (IGM), has announced that this procedure has been adopted as the standard diagnostic test for Cystic Fibrosis. A multiplexing protocol on a 316 chips allows to lower the cost of the analysis of a DNA samples at $200. This analysis looks at the 1,000 most relevant mutations identified so far providing an 98% detection rate, a remarkable improvement over the 88% obtained with the previous protocol. IGM also offers a complete mitochondrial DNA analysis for about $500 and revealed the intention to expand the NGS based diagnostic protocols to other frequently asked tests.

The diagnostic applications also represent a top priority for the NGS leader companies. Illumina has recently announced its new TruSight kits for the MiSeq (see also a post on GenomeWeb) which are targeted to various genetic disease such as autism, cardiomyopathies and pediatric inherited diseases. To complete the offer they also include a sort of Exome kit that covers all the genes reported in the Human Gene Mutation Database. Four months ago Life Technologies has presented its AmpliSeq kit on Inherited disease and with the AmpliSeq Designer they provide a tool for customer tailored target resequencing. Both companies also sell solutions targeted to the analysis of cancer genes, which is another diagnostic area with a tremendous expansion.

For sure NGS has enormously boosted the development of sequencing applications for diagnostic testings as well as the discovery of novel functional variants, opening the path leading to the long waited Personal Genomics and Personal Medicine.

Tuesday, 11 September 2012

Flash Report: we certainly do not want to miss the avalanche of new data from the ENCODE Project

We are considering to ask for a sabbatical to read (and maybe trying to understand...) the coordinated set of 30 papers published a few days ago in multiple journals by the ENCODE Project.
In collaboration with Nature they even developed a nice tool (Nature ENCODE Explorer) to facilitate the identification of the paper you want to read.

Flash Report: Wanna win a complete Illumina MiSeq system?

You have time until October 15, 2012. You just need to fill out a form available at this address.
Interestingly, "no prior experience with next-generation sequencing is necessary".
Grants will be scored on three main criteria:
- Scientific Merit
- Originality
- Fit with MiSeq

What do you win?
Three grants will be awarded, each including:
- One MiSeq system
- Ten sequencing reagent kits
- Choice of sample prep (Nextera DNA Sample Prep or TruSeq Custom Amplicon)
- BaseSpace cloud computing analysis and 1TB of storage
- Avadis NGS, a biologist-friendly bioinformatics desktop tool

New NGS site: Genomics Law Report

We added the link to an interesting (and somehow unsettling) web site in the list of external resources of genomics news (it's on the right side of our blog page).

"The Genomics Law Report, a publication of the law firm Robinson, Bradshaw & Hinson, analyzes the legal implications of important developments in the fields of genomics and personalized medicine — including key litigation, legislative, regulatory and policymaking activities — to help make sense of these complex and rapidly changing areas of law and policy".

Friday, 7 September 2012

PubMed Highlight: NGS in molecular diagnosis of Usher syndrome

An increasing number of papers have been published showing how NGS technology could now be effectively applied in the diagnosis of various genetic syndromes. Through WES or target resequencing, this technology could be particularly useful in the case of diseases characterized by an heterogeneous genetic background. However, there still are some limitations, mainly due to the need of a high coverage for a confident call of genetic variants. This issue is discussed in this interesting paper reporting molecular diagnosis of Usher syndrome using 3 different NGS platforms.

PLoS One. 2012;7(8):e43799. Epub 2012 Aug 29.

Molecular diagnosis of usher syndrome: application of two different next generation sequencing-based procedures.


Cluster in Biomedicine (CBM) scrl - Genomics, Area Science Park, Basovizza, Trieste, Italy.


Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25×, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified.

Wednesday, 5 September 2012

PubMed Highlight: A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers

BMC Genomics. 2012 Jul 24;13:341.

A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.


Wellcome Trust Sanger Institute, Hinxton, UK.




Next generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent's PGM, Pacific Biosciences' RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy.


Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform.


All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.

Monday, 3 September 2012

A couple of interesting summer papers...

We are back from summer vacations. Here are a couple of papers published in August that are worth reading.
The first one is a study showing how NGS can be a useful and powerful method to improve diagnosis in syndromes with complex clinical presentation. The second paper illustrates the use of Solid and IonTorrent NGS platforms in diagnostic routine for the screening of BRCA mutations.

Exploring the utility of Whole-Exome Sequencing as a diagnostic tool in a child with Atypical Episodic Muscle Weakness.
Clin Genet. 2012 Aug 17

Hanchard NA, Murdock DR, Magoulas PL, Bainbridge M, Muzny D, Wu Y, Wang M, McGuire AL, Lupski JR, Gibbs RA, Brown CW.
Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, U.S.A.; Texas Children's Hospital, 6621 Fannin Street,, Houston, Texas, 77030, U.S.A.

The advent of whole-exome next-generation sequencing (WES) has been pivotal for the molecular characterization of Mendelian disease; however, the clinical applicability of WES has remained relatively unexplored. We describe our exploration of WES as a diagnostic tool in a three-year old female patient with a two-year history of episodic muscle weakness and paroxysmal dystonia who presented following a previous extensive but unrevealing diagnostic work-up. WES was performed on the proband and her two parents. Parental exome data was used to filter potential de novo genomic events in the proband and suspected variants were confirmed using di-deoxy sequencing. WES revealed a de novo non-synonymous mutation in exon 21 of the calcium channel gene CACNA1S that has been previously reported in a single patient as a rare cause of atypical hypokalemic periodic paralysis. This was unexpected, as the proband's original differential diagnosis had included hypokalemic periodic paralysis, but clinical and laboratory features were equivocal, and standard clinical molecular testing for hypokalemic periodic paralysis and related disorders was negative. This report highlights the potential diagnostic utility of WES in clinical practice, with implications for the approach to similar diagnostic dilemmas in the future.

Development of a Next-Generation Sequencing Method for BRCA Mutation Screening: A Comparison between a High-Throughput and a Benchtop Platform.
J Mol Diagn. 2012 Aug 22.

Chan M, Mo Ji S, Yeo ZX, Gan L, Yap E, Yap YS, Ng R, Tan PH, Ho GH, Ang P, Lee AS.
Division of Medical Sciences, National Cancer Centre, Singapore, Republic of Singapore.

In a clinical setting, next-generation sequencing (NGS) approaches for the enrichment and resequencing of DNA targets may have limitations in throughput, cost, or accuracy. We evaluated an NGS workflow for targeted DNA sequencing for mutation detection. Targeted sequence data of the BRCA1 and BRCA2 genes, generated using a PCR-based, multiplexed NGS approach using the SOLiD 4 (n = 24) and Ion Torrent PGM (n = 20) next-generation sequencers, were evaluated against sequence data obtained by Sanger sequencing. The overall sensitivity for SOLiD and PGM were 97.8% (95% CI = 94.7 to 100.0) and 98.9% (95% CI = 96.8 to 100.0) respectively, using automated variant calling. The specificity for the SOLiD platform was high, at 100.0% (95% CI = 99.3 to 100.0). PGM correctly identified all 3 indels, but 68 false-positive indels were also called. Equimolar normalization of amplicons was not necessary for successful NGS. Both platforms are highly amenable to scale-up (>384 or >16 samples per run for SOLiD and PGM, respectively), potentially reducing the reagent cost for BRCA testing to <US$200. Only 325 ng of DNA per patient is required, with similar coverage and accuracy obtained using DNA derived from peripheral blood or buccal wash samples. The strategy described is accurate and easy to incorporate into conventional workflow, and shows potential for mutation screening of clinically important gene targets in genetic disorders.

Revive and Restore...How to to bring extinct species back to life

I've recently read in a post on GenomeWeb this curious news about a new company called Revive and Restore. Their main goal is to use advanced genomic technologies to recover or reconstruct the DNA of extinct species with the aim to revive them.

In an interview posted at, Ryan Phelan, executive director of Revive and Restore, says the effort grew out of her work with George Church and the Personal Genome Project. "Right now, George's approach of basically editing the genome starts to make the concept of bringing something back really plausible," she says.
The idea is to recover genome sequence information from extinct species and then use it to massively edit the DNA of a still living close relative until its genome matches that of the extinct one.

Reading this news, one could not avoid to remember the famous Jurassic Park movie, and the unlucky ending of their project of reviving dinosaurs. Honestly, I prefer to see a T-rex standing in a museum rather than a living one hunting at me... Going back to the real world, this project rises major ethical and ecological issues. Who is the one who can decide which species to revive? What would be their impact on the ecosystem?
In fact exctinction is a consequence of natural selection, it's the way nature shapes the world we live in. However Phelan argues that extinction "is 99.9 percent caused by man," and if that's the case, "do we have a little bit of responsibility to think about bringing it back now that we have science that can easily allow for it?".

However, things are moving fast and a new project has started at Revive and Restore to recover the extinct Passenger Pidgeon, editing the genome of the common Pidgeon.