Do you cov me? Effect of coverage reduction on species identification and genome reconstruction in complex biological matrices by metagenome shotgun high-throughput sequencing

Shotgun metagenomics sequencing is a powerful tool for the characterization of complex biological matrices, enabling analysis of prokaryotic and eukaryotic organisms and viruses in a single experiment, with the possibility of reconstructing de novo the whole metagenome or a set of genes of interest. One of the main factors limiting the use of shotgun metagenomics on wide scale projects is the high cost associated with the approach. We set out to determine if it is possible to use shallow shotgun metagenomics to characterize complex biological matrices while reducing costs. We measured the variation of several summary statistics simulating a decrease in sequencing depth by randomly subsampling a number of reads. The main statistics that were compared are alpha diversity estimates, species abundance, and ability of reconstructing de novo the metagenome in terms of length and completeness. Our results show that diversity indices of complex prokaryotic, eukaryotic and viral communities can be accurately estimated with 500,000 reads or less, although particularly complex samples may require 1,000,000 reads. On the contrary, any task involving the reconstruction of the metagenome performed poorly, even with the largest simulated subsample (1,000,000 reads). The length of the reconstructed assembly was smaller than the length obtained with the full dataset, and the proportion of conserved genes that were identified in the meta-genome was drastically reduced compared to the full sample. Shallow shotgun metagenomics can be a useful tool to describe the structure of complex matrices, but it is not adequate to reconstruct—even partially—the metagenome

Metagenomic profiles of different types of Italian high-moisture Mozzarella cheese

The microbiota of different types of Italian high-moisture Mozzarella cheese produced using cow or buffalo milk, acidified with natural or selected cultures, and sampled at the dairy or at the mass market, was evaluated using a Next Generation Sequencing approach, in order to identify possible drivers of the bacterial diversity. Cow Mozzarella and buffalo Mozzarella acidified with commercial cultures were dominated by Streptococcus thermophilus, while buffalo samples acidified with natural whey cultures showed similar prevalence of L. delbrueckii subsp. bulgaricus, L. helveticus and S. thermophilus. Moreover, several species of non-starter lactic acid bacteria were frequently detected. The diversity in cow Mozzarella microbiota was much higher than that of water buffalo samples. Cluster analysis clearly separated cow's cheeses from buffalo's ones, the former having a higher prevalence of psychrophilic taxa, and the latter of Lactobacillus and Streptococcus. A higher prevalence of psychrophilic species and potential spoilers was observed in samples collected at the mass retail, suggesting that longer exposures to cooling temperatures and longer production-to-consumption times could significantly affect microbiota diversity. Our results could help in detecting some kind of thermal abuse during the production or storage of mozzarella cheese

Exome Capture with Heterologous Enrichment in Pig (Sus scrofa).

The discovery of new protein-coding DNA variants related to carcass traits is very important for the Italian pig industry, which requires heavy pigs with higher thickness of subcutaneous fat for Protected Designation of Origin (PDO) productions. Exome capture techniques offer the opportunity to focus on the regions of DNA potentially related to the gene and protein expression. In this research a human commercial target enrichment kit was used to evaluate its performances for pig exome capture and for the identification of DNA variants suitable for comparative analysis. Two pools of 30 pigs each, crosses of Italian Duroc X Large White (DU) and Commercial hybrid X Large White (HY), were used and NGS libraries were prepared with the SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library (Agilent). A total of 140.2 M and 162.5 M of raw reads were generated for DU and HY, respectively. Average coverage of all the exonic regions for Sus scrofa (ENSEMBL Sus_scrofa.Sscrofa10.2.73.gtf) was 89.33X for DU and 97.56X for HY; and 35% of aligned bases uniquely mapped to off-target regions. Comparison of sequencing data with the Sscrofa10.2 reference genome, after applying hard filtering criteria, revealed a total of 232,530 single nucleotide variants (SNVs) of which 20.6% mapped in exonic regions and 49.5% within intronic regions. The comparison of allele frequencies of 213 randomly selected SNVs from exome sequencing and the same SNVs analyzed with a Sequenom MassARRAY® system confirms that this "human-on-pig" approach offers new potentiality for the identification of DNA variants in protein-coding genes

Heart failure due to 'stress cardiomyopathy': a severe manifestation of the opioid withdrawal syndrome.

Takotsubo cardiomyopathy (TTC) is a transient left ventricular (LV) dysfunction due to akinesia of the LV mid-apical segments (‘apical ballooning’) in the absence of critical coronary stenoses which can be complicated in the acute phase by heart failure, mitral regurgitation, life-threatening ventricular arrhythmias, or apical LV thrombosis. The syndrome is typically precipitated by intense emotional or physical stress; however, other causes of sympathetic overstimulation including administration of exogenous sympathomimetics or withdrawal of sympathetic antagonists can trigger TTC. We report the case of a patient who unexpectedly developed an ‘apical ballooning’ with severe reduction in the LV systolic function and heart failure after the withdrawal of methadone. The case supports the concept that increased sympathetic activity secondary to opioids withdrawal can trigger a stress-induced severe LV dysfunction. Physicians should be aware that the abrupt discontinuation of a long-term therapy with opioids may lead to serious cardiac complications. The administration of clonidine may be considered to prevent early clinical manifestations of addictive withdrawal, including TTC

Apical ballooning with mid-ventricular obstruction: the many faces of Takotsubo cardiomyopathy.

Takotsubo cardiomyopathy (TTC) is a transient left ventricular dysfunction due to akinesia of the left-ventricular (LV) mid-apical segments (apical ballooning), which can cause severe reduction in LV systolic function. The typical clinical picture of TTC include chest pain, electrocardiographic changes consisting of mild ST-segment elevation followed by diffuse deep T-wave inversion, QTc interval prolongation and mild troponin release in the absence of significant coronary stenoses. The syndrome often affects post-menopausal women and is triggered by sympathetic overstimulation, like intense physical or emotional stress, so that it is called the “broken heart syndrome”. Although left-ventricular systolic dysfunction usually fully recovers within few days, heart failure can still complicate the early phase. We report a case of stress-induced cardiomyopathy that had full recovery after 4 weeks of follow up. The main electrocardiographic, angiographic and imaging features are discussed

Receiver operating characteristic (ROC) curve.

<p>An example of Receiver operating characteristic (ROC) analysis for the prediction of ‘true segregating SNVs’ in the HY population using the allele frequencies in the exome capture experiment at a MAF threshold of 0.15.</p

Visualization with Circos software of differences in allele frequencies between pool DU and pool HY of pigs.

<p>The spots within circle represent the SNVs mapped on each chromosome and the distance between the inner and the outer circles indicates the difference in frequency of the reference allele between DU and HY. Therefore, SNVs with a difference > +0.5 between crossbreeds are placed in the outer layer, whereas those SNVs with a difference < –0.5 are located in the inner layer. Variants with allelic difference included between ± 0.5 are reported in the intermediate layer.</p