Meraculous: De Novo Genome Assembly with Short Paired-End Reads

We describe a new algorithm, meraculous, for whole genome assembly of deep paired-end short reads, and apply it to the assembly of a dataset of paired 75-bp Illumina reads derived from the 15.4 megabase genome of the haploid yeast Pichia stipitis. More than 95% of the genome is recovered, with no errors; half the assembled sequence is in contigs longer than 101 kilobases and in scaffolds longer than 269 kilobases. Incorporating fosmid ends recovers entire chromosomes. Meraculous relies on an efficient and conservative traversal of the subgraph of the k-mer (deBruijn) graph of oligonucleotides with unique high quality extensions in the dataset, avoiding an explicit error correction step as used in other short-read assemblers. A novel memory-efficient hashing scheme is introduced. The resulting contigs are ordered and oriented using paired reads separated by ∼280 bp or ∼3.2 kbp, and many gaps between contigs can be closed using paired-end placements. Practical issues with the dataset are described, and prospects for assembling larger genomes are discussed

Constitutively Active Canonical NF-κB Pathway Induces Severe Bone Loss in Mice

Physiologic osteoclastogenesis entails activation of multiple signal transduction pathways distal to the cell membrane receptor RANK. However, atypical osteoclastogenesis driven by pro-inflammatory stimuli has been described. We have reported recently a novel mechanism whereby endogenous mutational activation of the classical NF-κB pathway is sufficient to induce RANKL/RANK-independent osteoclastogenesis. Here we investigate the physiologic relevance of this phenomenon in vivo. Using a knock-in approach, the active form of IKK2, namely IKK2SSEE, was introduced into the myeloid lineage with the aid of CD11b-cre mice. Phenotypic assessment revealed that expression of IKK2SSEE in the myeloid compartment induced significant bone loss in vivo. This observation was supported by a dramatic increase in the number and size of osteoclasts in trabecular regions, elevated levels of circulating TRACP-5b, and reduced bone volume. Mechanistically, we observed that IKK2SSEE induced high expression of not only p65 but also p52 and RelB; the latter two molecules are considered exclusive members of the alternative NF-κB pathway. Intriguingly, RelB and P52 were both required to mediate the osteoclastogenic effect of IKK2SSEE and co-expression of these two proteins was sufficient to recapitulate osteoclastogenesis in the absence of RANKL or IKK2SSEE. Furthermore, we found that NF-κB2/p100 is a potent inhibitor of IKK2SSEE-induced osteoclastogenesis. Deletion of p52 enabled more robust osteoclast formation by the active kinase. In summary, molecular activation of IKK2 may play a role in conditions of pathologic bone destruction, which may be refractory to therapeutic interventions targeting the proximal RANKL/RANK signal

Evaluation of Methods for De Novo Genome Assembly from High-Throughput Sequencing Reads Reveals Dependencies That Affect the Quality of the Results

Recent developments in high-throughput sequencing technology have made low-cost sequencing an attractive approach for many genome analysis tasks. Increasing read lengths, improving quality and the production of increasingly larger numbers of usable sequences per instrument-run continue to make whole-genome assembly an appealing target application. In this paper we evaluate the feasibility of de novo genome assembly from short reads (≤100 nucleotides) through a detailed study involving genomic sequences of various lengths and origin, in conjunction with several of the currently popular assembly programs. Our extensive analysis demonstrates that, in addition to sequencing coverage, attributes such as the architecture of the target genome, the identity of the used assembly program, the average read length and the observed sequencing error rates are powerful variables that affect the best achievable assembly of the target sequence in terms of size and correctness

Gene-Boosted Assembly of a Novel Bacterial Genome from Very Short Reads

Recent improvements in technology have made DNA sequencing dramatically faster and more efficient than ever before. The new technologies produce highly accurate sequences, but one drawback is that the most efficient technology produces the shortest read lengths. Short-read sequencing has been applied successfully to resequence the human genome and those of other species but not to whole-genome sequencing of novel organisms. Here we describe the sequencing and assembly of a novel clinical isolate of Pseudomonas aeruginosa, strain PAb1, using very short read technology. From 8,627,900 reads, each 33 nucleotides in length, we assembled the genome into one scaffold of 76 ordered contiguous sequences containing 6,290,005 nucleotides, including one contig spanning 512,638 nucleotides, plus an additional 436 unordered contigs containing 416,897 nucleotides. Our method includes a novel gene-boosting algorithm that uses amino acid sequences from predicted proteins to build a better assembly. This study demonstrates the feasibility of very short read sequencing for the sequencing of bacterial genomes, particularly those for which a related species has been sequenced previously, and expands the potential application of this new technology to most known prokaryotic species

Addressing challenges in the production and analysis of illumina sequencing data

Advances in DNA sequencing technologies have made it possible to generate large amounts of sequence data very rapidly and at substantially lower cost than capillary sequencing. These new technologies have specific characteristics and limitations that require either consideration during project design, or which must be addressed during data analysis. Specialist skills, both at the laboratory and the computational stages of project design and analysis, are crucial to the generation of high quality data from these new platforms. The Illumina sequencers (including the Genome Analyzers I/II/IIe/IIx and the new HiScan and HiSeq) represent a widely used platform providing parallel readout of several hundred million immobilized sequences using fluorescent-dye reversible-terminator chemistry. Sequencing library quality, sample handling, instrument settings and sequencing chemistry have a strong impact on sequencing run quality. The presence of adapter chimeras and adapter sequences at the end of short-insert molecules, as well as increased error rates and short read lengths complicate many computational analyses. We discuss here some of the factors that influence the frequency and severity of these problems and provide solutions for circumventing these. Further, we present a set of general principles for good analysis practice that enable problems with sequencing runs to be identified and dealt with

Early Clinical Manifestations Associated with Death from Visceral Leishmaniasis

The visceral leishmaniasis (VL) is a disease potentially fatal if not diagnosed and treated opportunely. This article presents the results of the study on the manifestations identified at the time of the clinical suspicion of the VL cases. This study was conducted in Belo Horizonte, the capital of the State of Minas Gerais, located in southeastern Brazil. This study is both timely and substantive because the Belo Horizonte is an area of transmission of VL, with one of the highest VL-death proportions of Brazil. The patients with higher risk of death had at least one of the following characteristics: ≥60 years, weakness, HIV co-infection, bleeding, jaundice and other associated infections. During the period 2002–2009, 8% to 22% of the patients with VL progressed to death in Belo Horizonte, whilst the proportion in the country was much lower and varied between 5% and 9%. This study has identified vulnerable patients who are at higher risk of death from VL and who would benefit from early predictive evaluation of the prognostic. Hence, the knowledge regarding the factors associated with death may contribute for clinical management and for reduction of deaths from VL

Evaluation of next-generation sequencing software in mapping and assembly

Next-generation high-throughput DNA sequencing technologies have advanced progressively in sequence-based genomic research and novel biological applications with the promise of sequencing DNA at unprecedented speed. These new non-Sanger-based technologies feature several advantages when compared with traditional sequencing methods in terms of higher sequencing speed, lower per run cost and higher accuracy. However, reads from next-generation sequencing (NGS) platforms, such as 454/Roche, ABI/SOLiD and Illumina/Solexa, are usually short, thereby restricting the applications of NGS platforms in genome assembly and annotation. We presented an overview of the challenges that these novel technologies meet and particularly illustrated various bioinformatics attempts on mapping and assembly for problem solving. We then compared the performance of several programs in these two fields, and further provided advices on selecting suitable tools for specific biological applications.published_or_final_versio

An evaluation of factors associated with taking and responding positive to the tuberculin skin test in individuals with HIV/AIDS

<p>Abstract</p> <p>Background</p> <p>The tuberculin skin test (TST) is still the standard test for detecting latent infection by <it>M tuberculosis </it>(LTBI). Given that the Brazilian Health Ministry recommends that the treatment of latent tuberculosis (LTBI) should be guided by the TST results, the present study sets out to describe the coverage of administering the TST in people living with HIV at two referral health centers in the city of Recife, where TST is offered to all patients. In addition, factors associated with the non-application of the test and with positive TST results were also analyzed.</p> <p>Methods</p> <p>A cross-sectional study was carried out with HIV patients, aged 18 years or over, attending outpatient clinics at the Correia Picanço Hospital/SES/PE and the Oswaldo Cruz/UPE University Hospital, who had been recommended to take the TST, in the period between November 2007 and February 2010. Univariate and multivariate logistic regression analyses were carried out to establish associations between the dependent variable - taking the TST (yes/no), at a first stage analysis, and the independent variables, followed by a second stage analysis considering a positive TST as the dependent variable. The odds ratio was calculated as the measure of association and the confidence interval (CI) at 95% as the measure of accuracy of the estimate.</p> <p>Results</p> <p>Of the 2,290 patients recruited, 1087 (47.5%) took the TST. Of the 1,087 patients who took the tuberculin skin test, the prevalence of TST ≥ 5 mm was 21.6% among patients with CD4 ≥ 200 and 9.49% among those with CD4 < 200 (p = 0.002). The patients most likely not to take the test were: men, people aged under 39 years, people with low educational levels and crack users. The risk for not taking the TST was statiscally different for health service. Patients who presented better immunity (CD4 ≥ 200) were more than two and a half times more likely to test positive that those with higher levels of immunodeficiency (CD4 < 200).</p> <p>Conclusions</p> <p>Considering that the TST is recommended by the Brazilian health authorities, coverage for taking the test was very low. The most serious implication of this is that LTBI treatment was not carried out for the unidentified TST-positive patients, who may consequently go on to develop TB and eventually die.</p