Determining the quality and complexity of next-generation sequencing data without a reference genome

We describe an open-source kPAL package that facilitates an alignment-free assessment of the quality and comparability of sequencing datasets by analyzing k-mer frequencies. We show that kPAL can detect technical artefacts such as high duplication rates, library chimeras, contamination and differences in library preparation protocols. kPAL also successfully captures the complexity and diversity of microbiomes and provides a powerful means to study changes in microbial communities. Together, these features make kPAL an attractive and broadly applicable tool to determine the quality and comparability of sequence libraries even in the absence of a reference sequence. kPAL is freely available at https://github.com/LUMC/kPAL.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0555-3) contains supplementary material, which is available to authorized users.     

Household Transmission of Influenza A(H1N1)pdm09 in the Pandemic and Post-Pandemic Seasons

Background

The transmission of influenza viruses occurs person to person and is facilitated by contacts within enclosed environments such as households. The aim of this study was to evaluate secondary attack rates and factors associated with household transmission of laboratory-confirmed influenza A(H1N1)pdm09 in the pandemic and post-pandemic seasons.

Methods

During the 2009–2010 and 2010–2011 influenza seasons, 76 sentinel physicians in Navarra, Spain, took nasopharyngeal and pharyngeal swabs from patients diagnosed with influenza-like illness. A trained nurse telephoned households of those patients who were laboratory-confirmed for influenza A(H1N1)pdm09 to ask about the symptoms, risk factors and vaccination status of each household member.

Results

In the 405 households with a patient laboratory-confirmed for influenza A(H1N1)pdm09, 977 susceptible contacts were identified; 16% of them (95% CI 14–19%) presented influenza-like illness and were considered as secondary cases. The secondary attack rate was 14% in 2009–2010 and 19% in the 2010–2011 season (p = 0.049), an increase that mainly affected persons with major chronic conditions. In the multivariate logistic regression analysis, the risk of being a secondary case was higher in the 2010–2011 season than in the 2009–2010 season (adjusted odds ratio: 1.72; 95% CI 1.17–2.54), and in children under 5 years, with a decreasing risk in older contacts. Influenza vaccination was associated with lesser incidence of influenza-like illness near to statistical significance (adjusted odds ratio: 0.29; 95% CI 0.08–1.03).

Conclusion

The secondary attack rate in households was higher in the second season than in the first pandemic season. Children had a greater risk of infection. Preventive measures should be maintained in the second pandemic season, especially in high-risk persons.     

Characterization of an apoplastic basic thaumatin-like protein from recalcitrant chestnut seeds

Mature chestnut seeds, with one of the highest moisture contents described to date, accumulate certain defensive proteins at unusually elevated levels. In this work a major 23-kDa thaumatin-like protein, termed CsTL1, has been purified from mature chestnut ( Castanea sativa ) cotyledons. Amino acid sequencing and characterization of its full-length cDNA indicate that CsTL1 is synthesized as a preprotein with a signal peptide 22 amino acids in length. The mature protein contains 16 conserved cysteine residues presumably involved in disulfide bonding and has a high isoelectric point (ca. 9). Unlike most basic pathogenesis-related (PR) proteins, mature CsTL1 is localized to the extracellular matrix, as revealed by immunoelectron microscopy studies of cotyledonary cells. The isolated protein has in vitro antifungal activity against Trichoderma viride and Fusarium oxysporum and shows strong synergistic effects with CsCh1, the most abundant chestnut cotyledon endochitinase. Moreover, both CsTL1 and CsCh1 appear to be regulated in the same manner during seed development and germination. These observations, along with the recent finding of endoglucanase activity for some TL proteins, support the notion that CsTL1 and CsCh1 are part of a complex seed defensive system against microbial growth. Another possibility is that these, and probably other seed PR proteins, have antifreeze activity. Both functions would be particularly relevant for chestnut seeds given their remarkable moisture content at maturity.     

Floristic relationship between scrubland and grassland patches in the Mediterranean landscape of the Iberian Peninsula

In the rural Mediterranean landscape, mosaics of patches of sclerophyllous scrubland and semi-natural grasslands are frequent. The plant communities of these patches, which are physiognomically easy to recognise, are very heterogeneous. The objective of this paper is to determine whether the patches of scrub-grassland represent an integrated response unit of the vegetation with regard to the physical environment (climatic, geographical and edaphic factors) and human use, or whether, on the contrary, this is an independent response. In order to do this a total of 50 sampling sites where scrubland and grassland patches were in contact were studied along a 370 km E-W mesoclimatic gradient from central Spain to Portugal (Iberian Peninsula). Two distinct zones, the east and the west halves of the study area, were identified according to the plant communities. Within each of these zones, each type of patch responded to the previously mentioned factors, differentially and independently. This determined a general lack of inter-community association or floristic correlation between scrub and grassland plant communities in contact. The spatial association is a random process probably related to the particular human management realised on every scrubland and grassland patch. The scrub–grassland pattern, which is characteristic of the Mediterranean landscape, does not represent an integrated response unit of the vegetation to a given environment, but rather the sum of the independent responses to the environment of the two patches in contact.Nomenclature for taxa: T. G. Tutin et al. 1694–1980. Flora Europaea Cambridge University Press, Cambridge.     

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) expression is downregulated in poorly differentiated breast invasive ductal carcinoma

Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx) is the only known enzyme able to reduce lipid peroxides bound to cell membranes. Moreover it has been involved in apoptosis and can influence intracellular signaling. To investigate the possible relationship between PHGPx and human cancer we have quantified PHGPx expression levels by real-time quantitative PCR and immunohistochemistry in tissue samples of human breast invasive ductal carcinoma from 34 patients compared with their own controls of benign breast tissue. PHGPx expression levels were compared with the clinical and pathological data of these patients. The results showed that PHGPx expression levels are downregulated in poorly differentiated (grade 3) breast invasive ductal carcinoma (P = 0.0043). PHGPx expression levels decreased gradually with tumor grade from grade 1 to grade 3.We also found a downregulation of PHGPx in cases that showed p53 accumulation compared with cases without p53 immunostaining (P = 0.0011). PHGPx was also downregulated in cases without progesterone receptors (PR) immunostaining compared with cases with PR immunostaining (P = 0.0165). Grade 3, p53 immunostaining and absence of PR immunostaining are poor prognostic factors. These results suggest that PHGPx downregulation could be related with a poorer prognosis in breast invasive ductal carcinoma.     

Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.     

Pattern diversity analysis of a clearing in a Quercus cerris wood

Linear transects were used to examine the spatial structure of the vegetation in a clearing in a Quercus cerris wood in Central Italy. A grassland-margin-shrubland-woodland gradient was identified.Multivariate classification and ordination methods and pattern diversity analysis were used to detect both floristic variation and spatial organization. One of the transects shows a clear gradient of variation as a function of space, crossing the floristically and structurally most heterogeneous part of the clearing. For the detailed analysis of this transect a sectorization is achieved. Each sector is characterized according to its floristic types and to the internal spatial organization.The pieces of information derived from the different analyses were concordant. In particular, the validity of the floristic types in the qualification of the different forms of spatial organization is confirmed.     

A targeted metabolomic procedure for amino acid analysis in different biological specimens by ultra-high-performance liquid chromatography–tandem mass spectrometry

Introduction

Amino acid analysis in biological fluids is essential for the study of inborn errors of metabolism (IEM) and other diseases.

Objectives

Our aim was to develop a UPLC-MS/MS procedure for the analysis of 25 amino acids and identification of 17 related compounds.

Methods

Sample treatment conditions were optimized for plasma, urine, cerebrospinal fluid (CSF) and dried blood spots. Amino acids and related compounds were analyzed on a Waters ACQUITY UPLC H-class instrument with a reversed-phase C-18 column using water and acetonitrile with 0.1% formic acid as the mobile phases (run time?=?9 min). The detection was performed with a Waters Xevo TQD triple-quadrupole mass spectrometer using positive electrospray ionization in the multiple reaction monitoring mode.

Results

The method linearity, intra-assay and inter-assay precision, detection limit, quantification limit and trueness analysis displayed adequate results in both physiological and pathological conditions. Method comparison was performed between UPLC-MS/MS and ion exchange chromatography (IEC) with ninhydrin derivatization, and the methods showed good agreement, except for 4-hydroxyproline, aspartate and citrulline. Paediatrics age-related reference values in plasma, urine and CSF were established and patients with different IEM were easily identified.

Conclusion

We report a modified UPLC-MS/MS procedure for the analysis of 42 amino acids and related compounds in different specimens. The method is fast, sensitive and robust, and it has been validated to be an alternative to the traditional IEC procedure as the routine method used in metabolic laboratories. The method greatly decreases the run time of the analysis while displaying good metrological results.