Friday, 27 July 2012

NGS PubMed Highlights: identification of CFTR modifier gene through exome sequencing

According to the authors "this is the first study to discover a gene for a complex trait, or at minimum a genetic modifier of a Mendelian trait, using exome sequencing and an extreme phenotype study design".

Nat Genet. 2012 Jul 8. doi: 10.1038/ng.2344.

Exome sequencing of extreme phenotypes identifies DCTN4 as a modifier of chronic Pseudomonas aeruginosa infection in cystic fibrosis.


Department of Biostatistics, University of Washington, Seattle, Washington, USA.


Exome sequencing has become a powerful and effective strategy for the discovery of genes underlying Mendelian disorders. However, use of exome sequencing to identify variants associated with complex traits has been more challenging, partly because the sample sizes needed for adequate power may be very large. One strategy to increase efficiency is to sequence individuals who are at both ends of a phenotype distribution (those with extreme phenotypes). Because the frequency of alleles that contribute to the trait are enriched in one or both phenotype extremes, a modest sample size can potentially be used to identify novel candidate genes and/or alleles. As part of the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP), we used an extreme phenotype study design to discover that variants in DCTN4, encoding a dynactin protein, are associated with time to first P. aeruginosa airway infection, chronic P. aeruginosa infection and mucoid P. aeruginosa in individuals with cystic fibrosis.

Thursday, 26 July 2012

Ion Torrent is the First Entrant in Genomics X Prize Competition

The Archon Genomics X Prize will award $10 million to the first team that sequences the complete genomes of 100 people in 30 days or less, for no more than $1,000 each, and with an error rate of no more than 0.0001 percent. The contest, announced in 2006, has now its first entrant: Life Technologies Corp.'s Ion Torrent, which on Monday said it was entering the fray. Kevin Davies covered the story for Bio-IT World. A commentary has also been published in Nature.

New comparisons on NGS-Platforms!

NGS is moving fast as usual: Ion Torrent has just released its new datasets generated with the Cancer Panel and Inherited Disease kits... Illumina is going to update its MiSeq technology to 400bp reads... While waiting for future developments take a look at these two papers. The second one is a review from the BGI team on their sequencing experience, with a comparison of the current technologies.


A tale of three next generation sequencing platforms: comparison of Ion torrent, pacific biosciences and illumina MiSeq sequencers.

BMC Genomics. 2012 Jul 24;13(1):341. [Epub ahead of print]

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-neutral and 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.


Comparison of next-generation sequencing systems.


NGS Sequencing Department, Beijing Genomics Institute (BGI), 4th Floor, Building 11, Beishan Industrial Zone, Yantian District, Guangdong, Shenzhen 518083, China.

J Biomed Biotechnol. 2012;2012:251364. Epub 2012 Jul 5.
With fast development and wide applications of next-generation sequencing (NGS) technologies, genomic sequence information is within reach to aid the achievement of goals to decode life mysteries, make better crops, detect pathogens, and improve life qualities. NGS systems are typically represented by SOLiD/Ion Torrent PGM from Life Sciences, Genome Analyzer/HiSeq 2000/MiSeq from Illumina, and GS FLX Titanium/GS Junior from Roche. Beijing Genomics Institute (BGI), which possesses the world's biggest sequencing capacity, has multiple NGS systems including 137 HiSeq 2000, 27 SOLiD, one Ion Torrent PGM, one MiSeq, and one 454 sequencer. We have accumulated extensive experience in sample handling, sequencing, and bioinformatics analysis. In this paper, technologies of these systems are reviewed, and first-hand data from extensive experience is summarized and analyzed to discuss the advantages and specifics associated with each sequencing system. At last, applications of NGS are summarized.

Friday, 20 July 2012

PubMed Highlight: Genome-wide Single-Cell Analysis of Recombination Activity and De Novo Mutation Rates in Human Sperm

Cell, Volume 150, Issue 2, 402-412, 20 July 2012; 10.1016/j.cell.2012.06.030

Jianbin Wang, H. Christina Fan, Barry Behr, Stephen R. Quake

- A microfluidic system for high-throughput single cell whole-genome amplification
- Personal recombination map by single-sperm whole-genome analysis
- Genome-wide meiotic drive and gene conversion tests
- De novo germline mutations with distinct molecular characteristics

Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual human’s genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individual’s gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.

Wednesday, 18 July 2012

Crowd-funded sequencing

Even if the cost of DNA sequencing is falling rapidly, it's still unaffordable to sustain WES for every single subject with a rare syndrome but without an obvious molecular diagnosis. As usual with rare disorders, family find difficulties even in engage in research project, since a single case pathology don't create enough interest.
Families remain stucked with a close relative suffering a debilitant condition and the idea that the only thing preventing them from getting the diagnosis is money. Luckly sometimes people can surprise you with generosity. With internet technology you can reach thousands of people in an instant and provide the sum needed for a WES became really easy.
This is called crowd-funding and this story of a 4-year-old child sequenced and apparently (results have not yet been released) diagnosed by WES analysis paied with people donations shows that it can be an effective solution. In fact also some associations use this method to provide sequencing to family with a relative affected by an undiagnosed rare syndrome. Even if this approach raise some concerns on genome data privacy and scientific testing of the results, it is good to see what good people can do together even in the sequencing field.

Tuesday, 17 July 2012

NGS PubMed highlights: Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells

Nature. 2012 Jul 11;487(7406):190-5. doi: 10.1038/nature11236.

Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells.


Complete Genomics, Inc., 2071 Stierlin Court, Mountain View, California 94043, USA.


Recent advances in whole-genome sequencing have brought the vision of personal genomics and genomic medicine closer to reality. However, current methods lack clinical accuracy and the ability to describe the context (haplotypes) in which genome variants co-occur in a cost-effective manner. Here we describe a low-cost DNA sequencing and haplotyping process, long fragment read (LFR) technology, which is similar to sequencing long single DNA molecules without cloning or separation of metaphase chromosomes. In this study, ten LFR libraries were made using only ∼100 picograms of human DNA per sample. Up to 97% of the heterozygous single nucleotide variants were assembled into long haplotype contigs. Removal of false positive single nucleotide variants not phased by multiple LFR haplotypes resulted in a final genome error rate of 1 in 10 megabases. Cost-effective and accurate genome sequencing and haplotyping from 10-20 human cells, as demonstrated here, will enable comprehensive genetic studies and diverse clinical applications.

[PubMed - in process]
[Available on 2013/1/12]

The miRNA that can burn you!

This is not strictly a genomics topic, but it's summer time and for me this means hot sun and sunburns... So it's hard to ignore this paper from Nature Medicine that casts light on why a funny day at the beach can results in a couple of day of pain (at least for me when I forget my sun protection lotion)... Authors show that all the pain comes from a miRNA (these little molecules play a role really everywhere!) that is sensitive to UV-B exposure. Nearby cells sense the damaged miRNA and trigger inflammatory response. Really a hot topic!

Ultraviolet radiation damages self noncoding RNA and is detected by TLR3.


Exposure to ultraviolet B (UVB) radiation from the sun can result in sunburn, premature aging and carcinogenesis, but the mechanism responsible for acute inflammation of the skin is not well understood. Here we show that RNA is released from keratinocytes after UVB exposure and that this stimulates production of the inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) from nonirradiated keratinocytes and peripheral blood mononuclear cells (PBMCs). Whole-transcriptome sequencing revealed that UVB irradiation of keratinocytes induced alterations in the double-stranded domains of some noncoding RNAs. We found that this UVB-damaged RNA was sufficient to induce cytokine production from nonirradiated cells, as UVB irradiation of a purified noncoding RNA (U1 RNA) reproduced the same response as the one we observed to UVB-damaged keratinocytes. The responses to both UVB-damaged self-RNAs and UVB-damaged keratinocytes were dependent on Toll-like receptor 3 (TLR3) and Toll-like receptor adaptor molecule 1 (TRIF). In response to UVB exposure, Tlr3(-/-) mice did not upregulate TNF-α in the skin. Moreover, TLR3 was also necessary for UVB-radiation-induced immune suppression. These findings establish that UVB damage is detected byTLR3 and that self-RNA is a damage-associated molecular pattern that serves as an endogenous signal of solar injury.

Tuesday, 10 July 2012

NGS can find the cause of your disease, then you have to find the money to cure it

The New York Times reports the instructive story of Dr. Lukas Wartman, a young talented medical oncologist, bone marrow transplant specialist, who developed leukemia himself. Genetics researchers at Washington University decided to sequence the genes (WGS? WES?) of cancer cells and healthy cells of their colleague but it was it RNA-seq that allowed to find the over expression of FLT3 in the leukemia cells. The gene's normal role is to make cells grow and proliferate, thus an overactive FLT3 might be making Wartman’s cancer cells multiply out of control. Luckily enough, there is a drug, Sutent, approved for treating advanced kidney cancer, that inhibits FLT3. The only problem was that the drug costs $330 a day, and Wartman’s insurance company would not pay for it.
You can read the (happy) end of the story in this New York Times article.

Photo: Dilip Vishwanat, NYT / NYTNS

Friday, 6 July 2012

Exome Sequencing in Neurology

In the recent June issue of Neurology journal two reviews draw our attention on the big steps made by exome sequencing in both clinical and translational research. Developments of NGS are running at a very high speed, and, if until some months ago the application of these techniques in clinic was still considered "potential", we have now the first evidences that "potential" is an adjective of the past. The review of Coppola and Geschwind discusses three milestone papers describing the diagnosis of neurological diseases using exome sequencing. One paper by Landourè et al. describes a novel mutation in the TRPV4 gene causing Charcot-Marie-Tooth 2C phenotype. In the manuscript of Sailer et al. exome sequencing was succesfully performed to identify a novel mutation in the PRKCG gene causing Spinocerebellar Ataxia 14 (SCA14). In the third study Pierson et al. identified 2 compound heterozygous mutations in GLB1, responsible for recessive juvenile-onset GM1 in a family where initial galactosidase enzyme analysis was reported as normal.
In the same issue of Neurology, a second review by Doherty and Bamshad cites a paper in which a variant of the TYK2 gene was discovered by exome sequencing in 4 individuals with Multiple Sclerosis.

Monday, 2 July 2012

Yak genome revealed

The yak is a large, long-haired bovine living in the Himalayan region, perfectly adapted to the extreme altitude and low temperatures. As many other organism living in extreme conditions, it's interesting to speculate which adaptative tricks it has evolved to survive in such an hostile environment. The paper from Qiu and collaborators provide us with the genome and all the answers!

From the paper:
"Genomic comparisons between yak and cattle identify an expansion in yak of gene families related to sensory perception and energy metabolism, as well as an enrichment of protein domains involved in sensing the extracellular environment and hypoxic stress. Positively selected and rapidly evolving genes in the yak lineage are also found to be significantly enriched in functional categories and pathways related to hypoxia and nutrition metabolism"