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

specific targeted integration of kanamycin resistance associated nonselectable dna in the genome of the yeast saccharomyces cerevisiae

ABSTRACT Sophisticated genome manipulation re-quires the possibility to modify any inter-genic or intragenic DNA sequence at will,without leaving large amounts of undesiredvector DNA at the site of alteration. To thisend, a series of vectors was developed froma previous gene knockout plasmid system tointegrate nonselectable foreign DNA at anydesired genomic location in yeast, with aminimum amount of residual plasmid DNA.These vectors have two mutated Flp recog-nition targets ( FRT ) sequences flanking the KanMX4 gene and multiple sites for sub-cloning the DNA fragment to be integrated.The selectable marker can be recycled byFlp site-specific excision between the iden-tical FRT s, thereby allowing the integrationof further DNA fragments. With this system,the NLS-tetR-GFP and DsRed genes weresuccessfully integrated at the thr1 locus,and the RVB1 gene was tagged at the C-terminus with the V5-epitope-6-histidinetag. This plasmid system provides for a newmolecular tool to integrate any DNA frag-ment at any genome location in

Environmental DNA: The First Snapshot of the Vertebrate Biodiversity in Three Sicilian Lakes

Freshwater ecosystems play a key role in global diversity and are subject to a series of anthropic impacts, often leading to biodiversity loss. The organisms inhabiting these sites continuously release DNA into the environment through cells, excrement, gametes and/or decomposing matter; thus, evaluation of this eDNA could revolutionize the monitoring of biodiversity. In this study, environmental DNA metabarcoding was used for the first time in three Sicilian lakes: Lake Poma, Piana degli Albanesi Lake and Lake Scanzano. Results obtained provide the first snapshot of vertebrate biodiversity in these three lakes, where little is known, to provide valuable information useful for creating a baseline of knowledge regarding the biodiversity in these three lakes. Another important result was the detection of marine species, most likely due to some kind of anthropogenic contamination. Environmental DNA is a useful tool to evaluate both biodiversity and the ecological status of the environment; it has the potential to complement traditional methods, and the use of both approaches may offer a more comprehensive understanding of the ecosystem

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

Identification of tumorigenesis-related mRNAs associated with RNA-binding protein HuR in thyroid cancer cells

RNA binding proteins (RBPs) play a central role in cell physiology and pathology. Among them, HuR is a nuclear RBP, which shuttles to the cytoplasm to allow its RNA targets processing. HuR over-expression and delocalization are often associated to cell transformation. Numerous cancers display increased HuR protein levels and its high cytoplasmic levels has been associated with a worse prognosis.In our study, we first evaluated HuR expression in normal and cancer thyroid tissues and then evaluated its function in thyroid cell lines. HuR is over-expressed in all thyroid tumor tissues; high cytoplasmic levels are detected in all thyroid carcinomas. HuR silencing decreased cell viability and determined apoptotic cell death, in a non-tumorigenic (Nthy-ori-3.1) and a tumorigenic (BCPAP) thyroid cell line. Global transcriptome analysis indicated that HuR silencing, though having similar biological effects, induces distinct gene expression modifications in the two cell lines. By using the RIP-seq approach, the HuR-bound RNA profiles of different thyroid cell lines were evaluated. We show that in distinct cell lines HuR-bound RNA profiles are different. A set of 114 HuR-bound RNAs distinguishing tumorigenic cell lines from the non-tumorigenic one was identified.Altogether, our data indicate that HuR plays a role in thyroid tumorigenesis. Moreover, our findings are a proof of concept that RBP targets differ between cells with the same origin but with distinct biological behavior

Functional and comparative characterization of Saccharomyces cerevisiae RVB1 and RVB2 genes with bacterial Ruv homologues

Expression of yeast RuvB-like gene analogues of bacterial RuvB is self-regulated, as episomal overexpression of RVB1 and RVB2 decreases the expression of their chromosomal copies by 85%. Heterozygosity for either gene correlates with lower double-strand break repair of inverted-repeat DNA and decreased survival after UV irradiation, suggesting their haploinsufficiency, while overexpression of the bacterial RuvAB complex improves UV survival in yeast. Rvb2p preferentially binds artificial DNA Holiday junctions like the bacterial RuvAB complex, whereas Rvb1p binds to duplex or cruciform DNA. As both proteins also interact with chromatin, their role in recombination and repair through chromatin remodelling, and their evolutionary relationship to the bacterial homologue, is discussed.Fil: Radovic, Slobodanka. Yeast Molecular Genetics Group; ItaliaFil: Rapisarda, Viviana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Yeast Molecular Genetics Group; ItaliaFil: Tosato, Valentina. Yeast Molecular Genetics Group; ItaliaFil: Bruschi, Carlo V.. Yeast Molecular Genetics Group; Itali

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

Growth fitness, heme uptake and genomic variants in mutants of oxygen-tolerant Lacticaseibacillus casei and Lactiplantibacillus plantarum strains

Adaptive Laboratory Evolution (ALE) is a powerful tool to improve the fitness of industrially relevant microorganisms, because it circumvents some of the problems related to the use of genetically modified strains. In this study, we used an ALE strategy involving serial batch cultivations in aerobic and respiratory conditions to generate spontaneous mutants from the respiration-competent strain Lacticaseibacillus casei N87. Genotypic changes in selected mutants were investigated using whole genome sequencing (WGS). The O2-tolerant Lactiplantibacillus plantarum C17 and its mutant C17-m58 (obtained from a previous ALE study) were included in heme uptake experiments and in WGS and variant calling analyses. Several Lcb. casei N87 mutants cultivated under aerobic and respiratory conditions showed improved biomass production, O2 uptake and oxidative stress tolerance compared to the parental strain. Mutants of Lcb. casei and Lpb. plantarum differed from the parental strains in the ability to use heme and menaquinone. High heme concentrations (> 10 mg/L), however, were toxic for all strains. Single nucleotide modifications (SNPs) were detected in some genes encoding for proteins and transcriptional regulators involved in carbon metabolism, oxidative stress, redox balance, and cell wall properties, but their role in the evolved phenotypes needs further investigations. We conclude that prolonged adaptation to aerobic and respiratory life-style may be used as natural strategy to generate strains with improved O2-consuming ability and oxidative stress tolerance, two important features to develop robust cultures and to reduce oxidative processes in foods

ENVIRONMENTAL DNA: A POSSIBLE TOOL TO EVALUATE THE VERTEBRATE BIODIVERSITY USING WATER SAMPLES OF THREE SICILIAN LAKE

Freshwater ecosystems play a key role for the biogeochemical cycles and are subject to several anthropic impact that cause a biodiversity loss. Conventional census methods seem to be insufficient and not very convenient for the detection of species. The organisms inhabiting these sites continuously release DNA, called "environmental DNA" (eDNA), through cells, excrement, gametes and/or decomposing material and its evaluation could revolutionize the monitoring of biodiversity (HARPER et al., 2019; SCHENEKAR, 2023). On the other hand, today there are considerable doubts about it: seems to be the non-existence of well-defined protocols which depend on the sampling site, environmental factors and extraction methods; degradation processes are very rapid and the costs of evaluations are very high (many samples are needed in different points). The aim of this study (part of RTDA-PON project) was to fine-tune the sampling and eDNA extraction protocol, reducing the costs and evaluating the potential of this method compared to the conventional methods. Another aim was evaluating the biodiversity of vertebrate in some Sicilian Lake for which until to date no more is known. In this study, the eDNA technique was used for the first time in three Sicilian lakes: Poma Lake, Piana degli Albanesi Lake and Scanzano Lake.The water sampled in each site was filtered and eDNA was extracted using extraction kit. Metabarcoding analysis was performed by IGA Technology Services s.r.l. The results showed the possibility to understand which types of phyla, class, order, family, gender, or species lives in these sites. The species not captured or not sighted using conventional methods were detected thanks to the use of eDNA and vice versa highlighting that the evaluation of eDNA can be useful to evaluate not only aquatic species but also other species that use these sites as a source of supply. Another important result was the possibility to detect DNA of species not typical of freshwater sites highlighting the contamination process. eDNA is a very important tool to evaluate the biodiversity and the health status of the environment, but on the other hand, for a more complete census, it is very important use also other conventional methods at the same times. Our preliminary results showed that eDNA method have the potential to replace the traditional methods of census but it is not yet the time to do it, using both methods is surely the best approach

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