Utilization of Probabilistic Models in Short Read Assembly from Second-Generation Sequencing

With the advent of cheaper and faster DNA sequencing technologies, assembly methods have greatly changed. Instead of outputting reads that are thousands of base pairs long, new sequencers parallelize the task by producing read lengths between 35 and 400 base pairs. Reconstructing an organism’s genome from these millions of reads is a computationally expensive task. Our algorithm solves this problem by organizing and indexing the reads using n-grams, which are short, fixed-length DNA sequences of length n. These n-grams are used to efficiently locate putative read joins, thereby eliminating the need to perform an exhaustive search over all possible read pairs. Our goal was develop a novel n-gram method for the assembly of genomes from next-generation sequencers. Specifically, a probabilistic, iterative approach was utilized to determine the most likely reads to join through development of a new metric that models the probability of any two arbitrary reads being joined together. Tests were run using simulated short read data based on randomly created genomes ranging in lengths from 10,000 to 100,000 nucleotides with 16 to 20x coverage. We were able to successfully re-assemble entire genomes up to 100,000 nucleotides in length.

A second generation radiation hybrid map to aid the assembly of the bovine genome sequence

BACKGROUND: Several approaches can be used to determine the order of loci on chromosomes and hence develop maps of the genome. However, all mapping approaches are prone to errors either arising from technical deficiencies or lack of statistical support to distinguish between alternative orders of loci. The accuracy of the genome maps could be improved, in principle, if information from different sources was combined to produce integrated maps. The publicly available bovine genomic sequence assembly with 6x coverage (Btau_2.0) is based on whole genome shotgun sequence data and limited mapping data however, it is recognised that this assembly is a draft that contains errors. Correcting the sequence assembly requires extensive additional mapping information to improve the reliability of the ordering of sequence scaffolds on chromosomes. The radiation hybrid (RH) map described here has been contributed to the international sequencing project to aid this process. RESULTS: An RH map for the 30 bovine chromosomes is presented. The map was built using the Roslin 3000-rad RH panel (BovGen RH map) and contains 3966 markers including 2473 new loci in addition to 262 amplified fragment-length polymorphisms (AFLP) and 1231 markers previously published with the first generation RH map. Sequences of the mapped loci were aligned with published bovine genome maps to identify inconsistencies. In addition to differences in the order of loci, several cases were observed where the chromosomal assignment of loci differed between maps. All the chromosome maps were aligned with the current 6x bovine assembly (Btau_2.0) and 2898 loci were unambiguously located in the bovine sequence. The order of loci on the RH map for BTA 5, 7, 16, 22, 25 and 29 differed substantially from the assembled bovine sequence. From the 2898 loci unambiguously identified in the bovine sequence assembly, 131 mapped to different chromosomes in the BovGen RH map. CONCLUSION: Alignment of the BovGen RH map with other published RH and genetic maps showed higher consistency in marker order and chromosome assignment than with the current 6x sequence assembly. This suggests that the bovine sequence assembly could be significantly improved by incorporating additional independent mapping information.

Nutritional Aspects of Second Generation Soy Foods

Samples of 15 second generation soy-based products (n = 3), commercially available, were analyzed for their protein and isoflavone contents and in vitro antioxidant activity, by means of the Folin-Ciocalteu reducing ability, DPPH radical scavenging capacity, and oxygen radical absorbance capacity. Isoflavone identification and quantification were performed by high-performance liquid chromatography. Products containing soy and/or soy-based ingredients represent important sources of protein in addition to the low fat amounts. However, a large variation in isoflavone content and in vitro antioxidant capacity was observed. The isoflavone content varied from 2.4 to 18.1 mg/100 g (FW), and soy kibe and soy sausage presented the highest amounts. Chocolate had the highest antioxidant capacity, but this fact was probably associated with the addition of cocoa liquor, a well-known source of polyphenolics. This study showed that the soy-based foods do not present a significant content of isoflavones when compared with the grain, and their in vitro antioxidant capacity is not related with these compounds but rather to the presence of other phenolics and synthetic antioxidants, such as sodium erythorbate. However, they may represent alternative sources and provide soy protein, isoflavones, and vegetable fat for those who are not ready to eat traditional soy foods.

Time to rehospitalization in patients with schizophrenia discharged on first generation antipsychotics, non-clozapine second generation antipsychotics, or clozapine

Rehospitalization is an important outcome of drug effectiveness in schizophrenia. In this study, the hypothesis that clozapine and some second generation antipsychotics (SGA) were superior to first generation antipsychotics (FGA) in preventing rehospitalization of patients with schizophrenia discharged from a university hospital in Brazil was tested. A retrospective observational study was conducted designed to evaluate time to rehospitalization of patients with schizophrenia discharged on a regimen of oral FGA, depot FGA, risperidone, olanzapine and amisulpride, other SGA, or clozapine, during a three-year follow-up period. Risk factors associated with rehospitalization were examined. Of the 464 patients with schizophrenia discharged from hospital, 242 met criteria for study entry. Higher rehospitalization rates were observed in patients treated with depot FGA (30%), risperidone (30%) and other SGA groups (28.5%), respectively. Clozapine was significantly associated with lower rehospitalization risk compared with risperidone. The risk of rehospitalization in patients on olanzapine and amisulpride, and oral FGA, was similar to that of patients in use of clozapine. These results however, are limited by the heterogeneity of illness severity across the groups. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Congenital Chagas disease of second generation in Santiago, Chile. Report of two cases

Congenital Chagas disease (CChD) has been reported in different countries, mostly in Latin America. In 1987 a fatal case of CChD of second generation (CChDSG) was published. Within a period of six months - 1989-1990 - two cases of CChDSG were diagnosed and studied in the city of Santiago. Two premature newborns, sons of two sisters, with moderate liver and spleen enlargement, were found to have positive serology for Chagas disease and xenodiagnoses. The mothers, urban residents all their lives, without antecedents of triatomine bugs contact or blood transfusions, showed positive serology and xenodiagnoses. Their mother (grandmother of the infants), lived 20 years in a Northern rural Chagas disease endemic locality, in a triatomine infested house. Afterwards, she moved to Santiago, where she married and has resided up to now. Serology and xenodiagnoses were also positive. All the Trypanosoma cruzi infected individuals were successfully treated with nifurtimox.

First- Versus Second-Generation Drug-Eluting Stents in Acute Coronary Syndromes (Katowice-Zabrze Registry)

Abstract Background: There are sparse data on the performance of different types of drug-eluting stents (DES) in acute and real-life setting. Objective: The aim of the study was to compare the safety and efficacy of first- versus second-generation DES in patients with acute coronary syndromes (ACS). Methods: This all-comer registry enrolled consecutive patients diagnosed with ACS and treated with percutaneous coronary intervention with the implantation of first- or second-generation DES in one-year follow-up. The primary efficacy endpoint was defined as major adverse cardiac and cerebrovascular event (MACCE), a composite of all-cause death, nonfatal myocardial infarction, target-vessel revascularization and stroke. The primary safety outcome was definite stent thrombosis (ST) at one year. Results: From the total of 1916 patients enrolled into the registry, 1328 patients were diagnosed with ACS. Of them, 426 were treated with first- and 902 with second-generation DES. There was no significant difference in the incidence of MACCE between two types of DES at one year. The rate of acute and subacute ST was higher in first- vs. second-generation DES (1.6% vs. 0.1%, p < 0.001, and 1.2% vs. 0.2%, p = 0.025, respectively), but there was no difference regarding late ST (0.7% vs. 0.2%, respectively, p = 0.18) and gastrointestinal bleeding (2.1% vs. 1.1%, p = 0.21). In Cox regression, first-generation DES was an independent predictor for cumulative ST (HR 3.29 [1.30-8.31], p = 0.01). Conclusions: In an all-comer registry of ACS, the one-year rate of MACCE was comparable in groups treated with first- and second-generation DES. The use of first-generation DES was associated with higher rates of acute and subacute ST and was an independent predictor of cumulative ST.

Next generation sequencing of pooled samples reveals new SNRNP200 mutations associated with retinitis pigmentosa.

The gene SNRNP200 is composed of 45 exons and encodes a protein essential for pre-mRNA splicing, the 200 kDa helicase hBrr2. Two mutations in SNRNP200 have recently been associated with autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease, in two families from China. In this work we analyzed the entire 35-Kb SNRNP200 genomic region in a cohort of 96 unrelated North American patients with adRP. To complete this large-scale sequencing project, we performed ultra high-throughput sequencing of pooled, untagged PCR products. We then validated the detected DNA changes by Sanger sequencing of individual samples from this cohort and from an additional one of 95 patients. One of the two previously known mutations (p.S1087L) was identified in 3 patients, while 4 new missense changes (p.R681C, p.R681H, p.V683L, p.Y689C) affecting highly conserved codons were identified in 6 unrelated individuals, indicating that the prevalence of SNRNP200-associated adRP is relatively high. We also took advantage of this research to evaluate the pool-and-sequence method, especially with respect to the generation of false positive and negative results. We conclude that, although this strategy can be adopted for rapid discovery of new disease-associated variants, it still requires extensive validation to be used in routine DNA screenings. © 2011 Wiley-Liss, Inc.

Next-generation sequencing of experimental mouse strains.

Since the turn of the century the complete genome sequence of just one mouse strain, C57BL/6J, has been available. Knowing the sequence of this strain has enabled large-scale forward genetic screens to be performed, the creation of an almost complete set of embryonic stem (ES) cell lines with targeted alleles for protein-coding genes, and the generation of a rich catalog of mouse genomic variation. However, many experiments that use other common laboratory mouse strains have been hindered by a lack of whole-genome sequence data for these strains. The last 5 years has witnessed a revolution in DNA sequencing technologies. Recently, these technologies have been used to expand the repertoire of fully sequenced mouse genomes. In this article we review the main findings of these studies and discuss how the sequence of mouse genomes is helping pave the way from sequence to phenotype. Finally, we discuss the prospects for using de novo assembly techniques to obtain high-quality assembled genome sequences of these laboratory mouse strains, and what advances in sequencing technologies may be required to achieve this goal.

Assessment of Epstein-Barr virus nucleic acids in gastric but not in breast cancer by next-generation sequencing of pooled Mexican samples

Gastric (GC) and breast (BrC) cancer are two of the most common and deadly tumours. Different lines of evidence suggest a possible causative role of viral infections for both GC and BrC. Wide genome sequencing (WGS) technologies allow searching for viral agents in tissues of patients with cancer. These technologies have already contributed to establish virus-cancer associations as well as to discovery new tumour viruses. The objective of this study was to document possible associations of viral infection with GC and BrC in Mexican patients. In order to gain idea about cost effective conditions of experimental sequencing, we first carried out an in silico simulation of WGS. The next-generation-platform IlluminaGallx was then used to sequence GC and BrC tumour samples. While we did not find viral sequences in tissues from BrC patients, multiple reads matching Epstein-Barr virus (EBV) sequences were found in GC tissues. An end-point polymerase chain reaction confirmed an enrichment of EBV sequences in one of the GC samples sequenced, validating the next-generation sequencing-bioinformatics pipeline.

Cultural self-identification(s) and personal social networks among first and second-generation immigrants

L’estudi examina les relacions entre (1) les xarxes socials personals de la població immigrant resident a Barcelona i (2) les seves identitats culturals múltiples. L’objectiu principal de l’estudi és entendre com el contingut i l’estructura de les relacions socials dels immigrants facilita o dificulta (1) tenir un sentiment de pertinença a les noves cultures d’acollida, la catalana i la espanyola, i (2) la integració d’aquestes noves identitats socioculturals amb la seva identitat d’origen en una nova identitat bicultural cohesiva. El nostre plantejament inicial era que els immigrants amb xarxes socials més diverses des del punt de vista de la seva composició cultural tindrien més recursos socials i experiències cognitives més diverses , factors que afavoreixen les identificacions múltiples i la participació cívica. Els resultats de l’estudi mostren que el grau d’identificació dels participants amb la seva cultura ètnica o d’origen és força alt i, en certa mesura, més alt en comparació amb les cultures d’acollida ( catalana, cívica i espanyola). Tanmateix, el vincle dels participants amb les cultures d’acollida (p. ex., la cultura catalana) és prou rellevant per a indicar una orientació bicultural (catalana i ètnica). Les anàlisis de correlacions revelen que sentir-se català no impedeix sentir-se part de la comunitat etnocultural d’origen. A més, existeix una interrelació entre l'orientació cultural catalana i la identificació amb les comunitats cíviques locals. De la mateixa manera, tenir competències en llengua catalana no va en detriment de les competències en llengua castellana. Les anàlisis també mostren que factors com l’orientació cultural catalana, l’ús del català i la identificació amb la cultura catalana tenen una correlació positiva amb el grau de chohesio de la indentitat bicultural, afavoreixen el benestar psicològic i disminueixen l’estrès aculturatiu. L’anàlisi de les xarxes socials mostra que la identificació amb la cultura catalana, l’orientació cultural catalana i la integració de la identitat són factors clau per tenir xarxes socials més diverses des del punt de vista ètnic i lingüístic, amb menys membres del col•lectiu d’origen, i amb subgrups o “cliques” culturalment més heterogenis. La identificació espanyola també prediu, en mesura més reduïda, la diversitat de les xarxes. Els nostres resultats contribueixen a la recerca actual i les teories sobre interculturalitat i identitat cultural.

Labeling Regulations and Segregation of First- and, Second-Generation Genetically Modified Products: Innovation Incentives and Welfare Effects, April 2005

We review some of the most significant issues and results on the economic effects of genetically modified (GM) product innovation, with emphasis on the question of GM labeling and the need for costly segregation and identity preservation activities. The analysis is organized around an explicit model that can accommodate the features of both first-generation and second-generation GM products. The model accounts for the proprietary nature of GM innovations and for the critical role of consumer preferences vis-à-vis GM products, as well as for the impacts of segregation and identity preservation and the effects of a mandatory GM labeling regulation. We also investigate briefly a novel question in this setting, the choice of “research direction”when both cost-reducing and quality-enhancing GM innovations are feasible.

Nonspecific PCR amplification by high-fidelity polymerases: implications for next-generation sequencing of AFLP markers.

High-fidelity 'proofreading' polymerases are often used in library construction for next-generation sequencing projects, in an effort to minimize errors in the resulting sequence data. The increased template fidelity of these polymerases can come at the cost of reduced template specificity, and library preparation methods based on the AFLP technique may be particularly susceptible. Here, we compare AFLP profiles generated with standard Taq and two versions of a high-fidelity polymerase. We find that Taq produces fewer and brighter peaks than high-fidelity polymerase, suggesting that Taq performs better at selectively amplifying templates that exactly match the primer sequences. Because the higher accuracy of proofreading polymerases remains important for sequencing applications, we suggest that it may be more effective to use alternative library preparation methods.

"Next-generation" Sequencing (NGS): The new genomic revolution

In the past 5 years "Next-generation" Sequencing (NGS) technologies have transformed genomics by delivering fast, inexpensive and accurate genomeinformation changing the way we think about scientific approaches in basic,applied and clinical research. The inexpensive production of large volumes ofsequence data is the main advantage over the automated Sanger method,making this new technology useful for many applications. In this chapter, a brieftechnical review of NGS technologies is given, along with the keys to NGSsuccess and a broad range of applications for NGS technologies.

Immunobiology of human papillomavirus infection and vaccination - implications for second generation vaccines.

Prophylactic human papillomavirus (HPV) L1 virus like particle (VLP) vaccines have been shown, in large clinical trials, to be very immunogenic, well-tolerated and highly efficacious against genital disease caused by the vaccine HPV types. However these vaccines, at the present, protect against only two of the 15 oncogenic genital HPV types, they are expensive, delivered by intramuscular injection and require a cold chain. The challenges are to develop cheap, thermo-stable vaccines that can be delivered by non-injectable methods that provide long term (decades) protection at mucosal surfaces to most, if not all, oncogenic HPV types that is as good as the current VLP vaccines. Current approaches include L1 capsomers, L2 protein and peptides, delivery via recombinant L1 bacterial and viral vectors and large-scale VLP production in plants. Rational design and successful development of such vaccines will be based on an understanding of the immune response, and particularly the 'cross talk' between the innate and adaptive responses. This will be central in the development of adjuvants and vaccine formulations that induce the response to provide effective protection.