Fire affects the taxonomic and functional composition of soil microbial communities,with cascading effects on grassland ecosystem functioning

Fire is a crucial event regulating the structure and functioning of many ecosystems. Yet few studies have focused on how fire affects taxonomic and functional diversities of soil microbial communities, along with changes in plant communities and soil carbon (C) and nitrogen (N) dynamics. Here, we analyze these effects in a grassland ecosystem 9 months after an experimental fire at the Jasper Ridge Global Change Experiment site in California, USA. Fire altered soil microbial communities considerably, with community assembly process analysis showing that environmental selection pressure was higher in burned sites. However, a small subset of highly connected taxa was able to withstand the disturbance. In addition, fire decreased the relative abundances of most functional genes associated with C degradation and N cycling, implicating a slowdown of microbial processes linked to soil C and N dynamics. In contrast, fire stimulated above‐ and belowground plant growth, likely enhancing plant–microbe competition for soil inorganic N, which was reduced by a factor of about 2. To synthesize those findings, we performed structural equation modeling, which showed that plants but not microbial communities were responsible for significantly higher soil respiration rates in burned sites. Together, our results demonstrate that fire ‘reboots’ the grassland ecosystem by differentially regulating plant and soil microbial communities, leading to significant changes in soil C and N dynamics.     

Functional analysis of RPS27 mutations and expression in melanoma

Next‐generation sequencing has enabled genetic and genomic characterization of melanoma to an unprecedent depth. However, the high mutational background plus the limited depth of coverage of whole‐genome sequencing performed on cutaneous melanoma samples make the identification of novel driver mutations difficult. We sought to explore the somatic mutation portfolio in exonic and gene regulatory regions in human melanoma samples, for which we performed targeted sequencing of tumors and matched germline DNA samples from 89 melanoma patients, identifying known and novel recurrent mutations. Two recurrent mutations found in the RPS27 promoter associated with decreased RPS27 mRNA levels in vitro. Data mining and IHC analyses revealed a bimodal pattern of RPS27 expression in melanoma, with RPS27‐low patients displaying worse prognosis. In vitro characterization of RPS27‐high and RPS27‐low melanoma cell lines, as well as loss‐of‐function experiments, demonstrated that high RPS27 status provides increased proliferative and invasive capacities, while low RPS27 confers survival advantage in low attachment and resistance to therapy. Additionally, we demonstrate that 10 other cancer types harbor bimodal RPS27 expression, and in those, similarly to melanoma, RPS27‐low expression associates with worse clinical outcomes. RPS27 promoter mutation could thus represent a mechanism of gene expression modulation in melanoma patients, which may have prognostic and predictive implications.     

Sequencing two Tyr::CreERT2 transgenic mouse lines

The Cre/loxP system is a powerful tool that has allowed the study of the effects of specific genes of interest in various biological settings. The Tyr::CreER^T2 system allows for the targeted expression and activity of the Cre enzyme in the melanocyte lineage following treatment with tamoxifen, thus providing spatial and temporal control of the expression of specific target genes. Two independent transgenic mouse models, each containing a Tyr::CreER^T2 transgene, have been generated and are widely used to study melanocyte transformation. In this study, we performed whole genome sequencing (WGS) on genomic DNA from the two Tyr::CreER^T2 mouse models and identified their sites of integration in the C57BL/6 genome. Based on these results, we designed PCR primers to accurately, and efficiently, genotype transgenic mice. Finally, we discussed some of the advantages of each transgenic mouse model.     

Analysis of microRNA expression profiles in porcine PBMCs after LPS stimulation

In the present study, we used microRNA (miRNA) sequencing to discover and explore the expression profiles of known and novel miRNAs in 1000 ng/ml LPS stimulated for 8 h vis-à-vis non-stimulated (i.e. control) PBMCs isolated from the blood of healthy pigs. A total of 291 known miRNAs were bio-computationally identified in porcine PBMCs, and 228 novel miRNAs (not enlisted in the swine mirBase) were identified. Among these miRNAs, ssc-miR-148a-3p, ssc-let-7g, ssc-let-7f, 3_8760, ssc-miR-26a, ssc-miR-451, ssc-miR-21, ssc-miR-30d, ssc-miR-99a and ssc-miR-103 were the top 10 most abundant miRNAs in porcine PBMCs. Through miRNA differential analysis combined with quantitative PCR, we found the expressions of ssc-miR-122, ssc-miR-129b, ssc-miR-17-5p and ssc-miR-152 were significantly changed in porcine PBMCs after LPS stimulation. Furthermore, targets prediction and function analysis indicated a significant enrichment in gene ontology functional categories related to diseases, immunity and inflammation. In conclusion, this study on profiling of miRNAs expressed in LPS-stimulated PBMCs provides an important reference point for future studies on regulatory roles of miRNAs in porcine immune system.     

Quantitative evaluation of intraspecific genetic diversity in a natural fish population using environmental DNA analysis

Recent advances in environmental DNA (eDNA) analysis using high‐throughput sequencing (HTS) enable evaluation of intraspecific genetic diversity in a population. As the intraspecific genetic diversity provides invaluable information for wildlife conservation and management, there is an increasing demand to apply eDNA analysis to population genetics and the phylogeography by quantitative evaluation of intraspecific diversity. However, quantitative evaluations of intraspecific genetic diversity using eDNA is not straightforward because the number of eDNA sequence reads obtained by HTS may not be an index of the quantity of eDNA. In this study, to quantitatively evaluate genetic diversity using eDNA analysis, we applied a quantitative eDNA metabarcoding method using the internal standard DNAs. We targeted Ayu (Plecoglossus altivelis altivelis) and added internal standard DNAs with known copy numbers to each eDNA sample obtained from three rivers during the library preparation process. The sequence reads of each Ayu haplotype were successfully converted to DNA copy numbers based on the relationship between the copy numbers and sequence reads of the internal standard DNAs. In all rivers, the calculated copy number of each haplotype showed a significant positive correlation with the haplotype frequency estimated by a capture‐based survey. Furthermore, estimates of genetic indicators such as nucleotide diversity based on the eDNA copy numbers were comparable with those estimated based on a capture‐based study. Our results demonstrate that eDNA analysis with internal standard DNAs enables reasonable quantification of intraspecific genetic diversity, and this method could thus be a promising tool in the field of population genetics and phylogeography.     

Evolutionary processes of melanomas from giant congenital melanocytic nevi

Melanoma can develop in a congenital melanocytic nevus (CMN). In fact, a large CMN is associated with a high risk of developing melanoma. Although melanomas arising from CMNs are thought to have a pathogenesis distinct from conventional melanomas, no studies have been conducted on the evolution or tumor heterogeneity of CMN melanomas. We applied multi‐region whole‐exome sequencing to investigate the clonal nature of driver events and evolutionary processes in CMNs and melanomas arising from CMNs. In two patients, we observed an independent subclonal evolution in cancerized fields of CMNs and chromosome 8q amplification in both melanomas arising from CMNs. The amplification of MYC, located in chromosome 8q, was correlated with the percentage of tumor cells expressing high levels of MYC protein detected in melanoma cells by immunohistochemistry. Our analysis suggests that each CMN cell may evolve sporadically and that amplification of MYC might be a key event for melanoma development in CMNs.     

Helping decision making for reliable and cost‐effective 2b‐RAD sequencing and genotyping analyses in non‐model species

High‐throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b‐RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b‐RAD protocols on non‐model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b‐RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade‐off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective‐base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b‐RAD protocols on non‐model organisms with different genome sizes, helping decision‐making for a reliable and cost‐effective genotyping.     

Index hopping on the Illumina HiseqX platform and its consequences for ancient DNA studies

The high‐throughput capacities of the Illumina sequencing platforms and the possibility to label samples individually have encouraged wide use of sample multiplexing. However, this practice results in read misassignment (usually <1%) across samples sequenced on the same lane. Alarmingly high rates of read misassignment of up to 10% were reported for lllumina sequencing machines with exclusion amplification chemistry. This may make use of these platforms prohibitive, particularly in studies that rely on low‐quantity and low‐quality samples, such as historical and archaeological specimens. Here, we use barcodes, short sequences that are ligated to both ends of the DNA insert, to directly quantify the rate of index hopping in 100‐year old museum‐preserved gorilla (Gorilla beringei) samples. Correcting for multiple sources of noise, we identify on average 0.470% of reads containing a hopped index. We show that sample‐specific quantity of misassigned reads depends on the number of reads that any given sample contributes to the total sequencing pool, so that samples with few sequenced reads receive the greatest proportion of misassigned reads. This particularly affects ancient DNA samples, as these frequently differ in their DNA quantity and endogenous content. Through simulations we show that even low rates of index hopping, as reported here, can lead to biases in ancient DNA studies when multiplexing samples with vastly different quantities of endogenous material.