454 Life Sciences

454 Ultra-Deep Sequencing Continues to Shed Light on HIV Drug-Resistance

In a study published this month in PLoS One, researchers at the Yale University School of Medicine have used the 454 Sequencing System to detect low-frequency drug-resistance mutations in antiretroviral-experienced patients with virologic failure. Previous studies (Simen et al. 2009, JID; Metzner et al. 2009, Clin Infect Dis) have shown that low-abundance viral variants (< 20%) often remain undetected with conventional genotyping technologies and that these variants can have significant impact on clinical outcomes, even at levels down to 1%. This most recent study compared ultra-deep sequencing using the GS FLX System and conventional Sanger sequencing to determine the prevalence of drug-resistance mutations in plasma samples from 22 antiretroviral-experienced subjects. The researchers found low-abundance drug-resistance associated mutations in all 22 subjects by 454 deep sequencing, but only in 3 subjects by Sanger sequencing. In all, conventional genotyping methods failed to detect 95% of the low-abundance drug resistance variants, whereas ultra-deep sequencing detected all mutations found by Sanger sequencing as well as the additional low-abundance variants. The latter variants correlated with the failing regimen in some cases, but more generally with antiretroviral regimens taken previously by the subject. Taking the low-frequency variants into account, the overall predicted burden of resistance was increase in 77% of the subjects. The authors added, "New sequencing technologies with better sensitivity such as ultra-deep pyrosequencing can provide important historical resistance information that help clinicians planning subsequent antiretroviral regimens for highly treatment-experienced patients, especially for those whose antiretroviral treatment histories and longitudinal HIV genotypes are not obtainable." Further investigations are needed to better understand the environments in which low-abundance drug-resistant variants develop, how they evolve and how they impact treatment response.

To read the study, click here.

Researchers Complete Draft Genome of Honey Bee Parasite with GS FLX System

A team of researchers from the US Department of Agriculture, the University of Maryland, Columbia University and 454 Life Sciences have published the first draft genome of Nosema ceranae, a honey bee microsporidian pathogen with links to Colony Collapse Disorder (CCD). During CCD, honeybee colonies inexplicably lose all of their worker bees. The phenomenon has resulted in a loss of 50-90% of colonies in beekeeping operations across the United States. Bees play an integral role in the world food supply and are essential for the pollination of more than 90 fruit and vegetable crops worldwide. The economic value of these agricultural products is placed at more than $14.6 billion in the United States alone.

While the causes of CCD are not yet known, and are likely to be caused by multiple factors, the Nosema ceranae fungal pathogen is one candidate disease agent. In order to understand the species and its effect on bees, the researchers sequenced and assembled the 7.86 million base genome with the Genome Sequencer FLX System. Sequencing analysis revealed that the genome contains about 2,614 protein-coding genes. With the complete genome assembly available, the researchers can now perform comparative analysis with genetic model organisms such as yeast and other related species. The new sequence information will ultimately aid the team in understanding the unknown, pathogenic elements of Nosema-honey bee interactions. The study results were published this month in PLoS Pathogens.

To read the study, click here.

454 Sequencing Enables De Novo Sequencing and Assembly of Rice Genome

A group of researchers at the University of Arizona, in collaboration with 454 Life Sciences, have demonstrated a strategy for de novo sequencing and assembly of the rice genome using a pooled BAC method with the Genome Sequencer FLX System. The ultimate aim of the project is to sequence the ten genome types of the genus Oryza, which range in size from 350 to 1,285 Mb and includes the world's most important food crop-rice. By sequencing the genomes of major food and energy crops, scientists hope to identify key genes and loci that correspond to plant growth in difficult conditions, such as less land, less water, poor soil, and fewer pesticides.

To accommodate the highly complex nature of the oryza genome, the researchers used a pooled BAC method with the GS FLX Titanium series long single reads (400 bp) and 3 Kb paired end reads. In a single pilot experiment the researchers generated a high quality draft sequence of an entire 18-Mb chromosome arm of O. barthii. They employed the GS Assembler and BABMUS software to assemble the arm into 44 scaffolds with an N50 scaffold size of 3.1 Mb and the largest scaffold size of 6.6 Mb. The assembly into a few large, scaffolds indicates the high quality draft produced with this method. The study concludes, "[The strategy] provides an immediate and practical solution to the rapid generation of genome sequences from large and complex eukaryotic genomes for accelerated biological discovery to address some of our most critical food and bioenergy challenges."

For the study abstract, click here.

Researchers Complete Mitochondrial Genome Sequence of the Extinct Tasmanian Tiger

A group of researchers, led by Stephan Schuster and Webb Miller at Pennsylvania State University, have reported the first two complete mitochondrial genome sequences of the thylacine, or Tasmanian tiger, which was declared extinct in 1936 upon death of the last captive animal. Much interest has been given to the Tasmanian tiger since it is phylogenetically isolated in its own taxonomic family, yet has a number of striking similarities to members of the placental carnivore family which includes wolves and dogs. This example of convergent evolution, or the acquisition of the same biological train in unrelated lineages, is of great interest to evolutionary biologists. The Tasmanian tiger has also received attention for its process of extinction and in discussions whether the species could be resurrected through ancient DNA research and modern reproductive medicine. In this study, the researchers used the Genome Sequencer FLX System and careful extraction techniques of ancient DNA from museum hair samples to sequence the complete mitochondrial genome. The researchers believe this method could be applied to a wide range of museum specimens, even for samples with a temperate storage history. By obtaining nuclear genome sequence data of extinct species, biologists will have the ability to observe evolutionary events directly rather than being limited to inferences based on samples from living organisms.

For the study abstract, click here.

454 Sequencing Reveals New Insights into Nerve-Dependent Limb Regeneration in Salamanders

Uncovering the molecular and genetic pathways involved in routine limb regeneration in salamanders has been the aim of research studies for hundreds of years. Recently, a group of researchers from the Spinal Cord and Brain Injury Research Center at the University of Kentucky and the Salk Institute for Biological Sciences used the Genome Sequencer FLX system to explore gene expression in Mexican axolotls with amputated forelimbs. While it has been widely demonstrated that salamander limb regeneration requires the presence of nerves, little research has taken a broad systems-level approach to understand why this occurs. Using cDNA sequencing with the GS FLX System the researchers compared transcript abundances among uninjured limbs, regenerating limbs, and limbs denervated at the time of amputation. By comparing gene expression across each of the salamander groups, the researchers were able characterize the downstream cellular events that are affected when nerve supply is removed from an amputated limb. They found that innervated and denervated limbs exhibit similar gene expression patterns for wound healing at five days post amputation but then diverged as a cell mass formed under the influence of nerves. These results are consistent with previous regeneration studies. One of the most exciting results of the study was the identification of thousands of novel gene sequences and functional pathways through deep cDNA sequencing. This new sequence information will greatly enrich future studies in wound healing, epigenetics, genome stability, and regeneration using the axolotl model.

For the study abstract, click here.

A complementary study employed BAC sequencing with the GS FLX System to assemble large regions of the salamander genome. The results show that axolotl genic regions encode novel genes and make a significant contribution to genome size. In fact, the researchers found that genes in the salamander genome are 5x larger than they are in humans and 2% more abundant. The study estimates the genic portion of the genome to be an astounding 2.8 gigabases.

For the study abstract click here

Transcriptome Sequencing Study Identifies Novel Prostate Cancer Gene Fusions

A team of researchers at the University of Michigan has used the Genome Sequencer FLX system to identify a number of novel gene fusions in prostate cancer by sequencing the transcriptomes of patient cell lines and tumor samples. Gene fusions refer to the merging of two previously separate genes during chromosomal rearrangements in cancer. In some cases, two gene-coding sequences are combined, leading to the expression of a fusion protein or abnormal expression of regulatory genes. While gene fusions are typically associated with rare blood, bone and soft-tissue cancers, recent studies have shown evidence of these mutations in solid tumors. In an initial proof of concept experiment, the researchers found that de novo discovery of gene fusions was impossible using only a short-read technology. Turning to the GS FLX System, the researchers created cDNA libraries of prostate cancer tumor and cell lines for sequencing. Using a combination of long and short reads, the long 454 reads provided the essential reference sequences for mapping candidate fusion genes. In the end, the researchers found a recurrent chimaeric transcript called SLC45A3-ELK4 and several fusions which may represent rare or private mutations. The results of the study, published in Nature, may lead to the development of new diagnostic markers and therapeutic targets for prostate cancer.

For the study abstract, click here.

Transcriptome sequencing uncovers breast cancer genomic rearrangements.

A team of researchers from the J. Craig Venter Institute and the Ludwig Institute for Cancer Research has used transcriptome sequencing with the 454 Sequencing system to discover novel genomic alterations that lead to gene fusions in a breast cancer cell line. Sequencing cDNA from a well-characterized breast cancer cell line, HCC1954, with the Genome Sequencer FLX System revealed 496 sequences that indicate chromosomal translocations. Of this total, the researchers confirmed six inter-chromosomal changes and one intra-chromosomal change that have the potential to affect the protein translating ability of at least nine genes. The team concluded that transcriptome sequencing with the 454 Sequencing system is very adept at finding genomic rearrangements and mutations associated with cancer. The results deepen our understanding of changes in the human genome that could be directly involved in cancer, leading the way for the development of new intervention approaches and improved patient outcomes.

For the study abstract, click here.

Researchers use GS FLX System for rapid sequencing of Avian Influenza H5N1 genomes

A team of researchers at the Institute of Diagnostic Virology in Germany have developed a simple and rapid method for preparing Avian Influenza H5N1 diagnostic samples for sequencing with the Genome Sequencer FLX system. The highly pathogenic avian influenza A virus (HPAIV) of subtype H5N1 has caused global concern as a potential pandemic threat, killing millions of poultry and crossing over to humans in a number of documented cases. In-depth full-length sequencing analysis of infected patient samples are needed immediately to determine the proper course of action after viral diagnosis. While traditional Sanger sequencing requires tedious cloning steps, 454 Sequencing enables unbiased sample preparation and reduces time from weeks to days. Using shotgun sequencing the method enables complete and accurate sequencing of multiple viral genomes within three days, directly from diagnostic samples. The speed of sequencing and quantity of viral genomes will only increase with the recent introduction of the GS FLX Titanium series reagents (not used for this study). The immediate availability of detailed sequencing information will allow researchers to take action against the spread of the virus and permit molecular epidemiology in an outbreak situation.

For the study abstract, click here.

CDC-led research team uses 454 Sequencing to discover a new Ebola virus linked to hemorrhagic fever outbreak in Uganda

In November 2007, a large hemorrhagic fever outbreak was reported in the townships of Bundibugyo and Kikyo in western Uganda. A total of 29 blood samples were gathered from infected patients and shipped to the Centers for Disease Control and Prevention (CDC) for testing. Evidence of acute ebolavirus infection, a known cause of hemorrhagic fever, was negative when initially tested with highly sensitive real-time PCR assays specific for Zaire and Sudan ebolaviruses. Using metagenomic analysis with the Genome Sequencer FLX System, researchers quickly identified the outbreak to be associated with a newly discovered ebolavirus species, named Bundibugyo ebolavirus. Sequencing of the complete viral genome suggests that the new virus is distantly related to the Cote d'Ivoire ebolavirus, the last new species of ebolavirus discovered 14 years ago. The study has important implications for design of future diagnostic assays to monitor hemorrhagic fever and efforts to develop effective treatments for this fatal disease.

To read the complete publication, click here

Resurrecting the Pleistocene: The genome of the extinct woolly mammoth

The era of high-throughput genomics continues to complement fossil records of extinct species, increasing our evolutionary understanding of modern-day relatives and possibly revealing the genetic factors that affected their extinction. Using the Genome Sequencer FLX System, researchers at Pennsylvania State University, led by Dr. Stephan Schuster, published in Nature 3.3 billion bases of the extinct woolly mammoth genome. Representing nearly 80% of the complete genome, it is the largest reported sequence of any extinct species to date. Analysis of the mammoth samples suggests one was male, and estimates their intra-species separation to be 1.5-2 million years. Comparative genomic analysis to modern-day elephants revealed that the species differ on average by one amino acid per protein, however which of these changes are functional important remains undetermined. Sequencing multiple genomes of both mammoth and elephant will enable future genome-wide population studies and will facilitate a direct observation of genetic changes in evolutionary time.

To read the New York Times cover story, click here

For the study abstract visit, click here

Study identifies a novel Borna virus as probable agent in fatal bird disease.

A team of researchers, led by Dr. Ian Lipkin at Columbia University, recently used high-throughput sequencing with the Genome Sequencer FLX system to characterize new strains of the Borna disease virus (BVD). The novel virus types were discovered in the genomes of three birds diagnosed with Proventricular dilatation disease (PDD), a common and deathly inflammatory syndrome infecting exotic avian species such as macaws and parrots. PDD is an infectious disease associated with rapid deterioration of the central, peripheral and autonomic nervous system. Similarly, the Borna disease virus is high neurotropic and has been described a wide variety of vertebrates including chickens, quails, rats, rabbits, cats, shrews and non human primates. Given their pathogenic similarity and conclusive genomic data, the study determines the bornaviruses in the birds is a plausible candidate causative agent of PDD. A testament to the speed and quality of the Genome Sequencer FLX System, the researchers conclude, "We are intrigued that whereas molecular discovery of the first BVD in the late 1980's required an investment of two years in subtractive cloning, high-throughput sequencing, bioinformatics and sequence databases enabled discovery of these two strains in two weeks." To read the full publication, click here

New virus threatening California grape vines is identified

Researchers in the laboratory of Dr. Adib Rowhani at the University of California in Davis have identified a novel RNA virus as the potential cause for declining Syrah grapevine cultivars. Recognizing the urgency to prevent continuing crop damage, the team, led by Dr. Maher Al Rwahnih, sequenced the entire RNA content of both diseased and an apparently healthy vine on the GS FLX System. Subsequent data analysis by GenomeQuest rapidly filtered through the enormous amounts of data and successfully identified a previously unknown virus. The combined sensitivity of 454 Sequencing and efficiency of GenomeQuest data analysis, allowed the researchers obtain the entire sequence of the viral genome and confirm its presence in unhealthy vines. The results of this research will potentially aid in faster identification and treatment of infected vines, contributing to better management of viral outbreaks. For more details on the study, click here

Metagenomics study reveals a single-species ecosystem deep within gold mine

An international team of researchers used metagenomic analysis with the GS FLX System to characterize the microbial diversity of water samples from deep within a South African gold mine. The results yielded a single bacteria, dubbed Candidatus Desulforudis audaxviator, composing >99.9% of the microbial community. Genomic analysis of the previously-unnamed microbe revealed genes involved in nitrogen and carbon fixation, suggesting that C. audaxviator is self-sufficient. The researchers concludes that the new organism is "capable of an independent life-style well suited to long-term isolation from photosphere deep within Earth's crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome." For the study abstract, click here

Researchers conclude circulating microRNAs are stable blood-based markers for cancer detection

A team of researchers in Washington state used the Genome Sequencer FLX System to demonstrate that circulating microRNAs (miRNAs) are promising blood-based markers for cancer classification and prognostication. MiRNAs are small regulatory RNAs, shown to be often misregulated in cancer. The recent study, published in PNAS, demonstrates in mice that miRNAs originating from a tumor enter the circulation in a stable form, where they can be readily detected and robustly distinguish animals with tumors from controls animals. The researchers extended their proof-of-principle experiment to cancer in humans, where detecting serum levels of a miRNA expressed in prostate cancer differentiated patients from healthy controls. The study´s results, achieved by sequencing small RNA libraries from human primary prostate cells with the 454 Genome Sequencer System, establish the blood-based measurement of tumor-derived miRNAs as a potential new approach for the detection of human cancer. For the study abstract, click here.

Study determines the bacterial diversity of chronic diabetic foot ulcers

In a recent study, published in PLoS ONE, researchers used 454 amplicon sequencing to evaluate the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. Determining the types of bacterial associated with these diabetics ulcers may ultimately enable the development of more effective treatment options. The study results showed that no one genera of bacteria was present in all 40 samples, suggesting that wound types may be defined by clusters of bacterial species rather than one type. The researchers termed these common bacterial groups "functional equivalent pathogroups" and hypothesize that the symbiotic nature of these bacterial contributes to the chronicity of diabetic foot wounds. The study also found that traditional clinical microbiology lab methods for bacteria detection dramatically overestimate the importance of organisms that are easily cultured. The authors called for a shift in methodology towards the molecular sequencing approaches such as bTEFAP using 454 Sequencing that unambiguously detect the bacterial diversity within samples. For the study abstract, click here