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Recent advances in sequencing technologies now permit the analyses of plant DNA from fossil samples (ancient plant DNA, plant aDNA), and thus enable the molecular reconstruction of palaeofloras.Hitherto, ancient frozen soils have proved excellent in preservingDNAmolecules, and have thus been the most commonly used source of plant aDNA. However, DNA from soil mainly represents taxa growing a fewmetres fromthe sampling point. Lakes have larger catchment areas and recent studies have suggested that plant aDNAfromlake sediments is a more powerful tool for palaeofloristic reconstruction. Furthermore, lakes can be found globally in nearly all environments, and are therefore not limited to perennially frozen areas. Here,we review the latest approaches and methods for the study of plant aDNA from lake sediments and discuss the progressmade up to the present.Weargue that aDNAanalyses add newand additional perspectives for the study of ancient plant populations and, in time, will provide higher taxonomic resolution and more precise estimation of abundance. Despite this, key questions and challenges remain for such plant aDNA studies. Finally, we provide guidelines on technical issues, including lake selection, and we suggest directions for future research on plant aDNA studies in lake sediments.

High‐throughput sequencing of the 16S rRNA gene on the Illumina platform is commonly used to assess microbial diversity in environmental samples. The MiniSeq, Illumina's latest benchtop sequencer, enables more cost‐efficient DNA sequencing relative to larger Illumina sequencing platforms (e.g., MiSeq). Here we used a modified custom primer sequencing approach to test the fidelity of the MiniSeq for high‐throughput sequencing of the V4 hypervariable region of 16S rRNA genes from complex communities in environmental samples. To this end, we designed additional sequencing primers that enabled application of a dual‐index barcoding method on the MiniSeq. A mock community was sequenced alongside the environmental samples in four different sequencing runs as a quality control benchmark. We were able to recapture a realistic richness of the mock community in all sequencing runs, and identify meaningful differences in alpha and beta diversity in the environmental samples. Furthermore, rarefaction analysis indicated diversity in many environmental samples was close to saturation. These results show that the MiniSeq can produce similar quantities of high‐quality V4 reads compared to the MiSeq, yet is a cost‐effective option for any laboratory interested in performing high‐throughput 16S rRNA gene sequencing. Innovation in next‐generation DNA sequencing technology continues to contribute to the democratization of 16S rRNA gene sequencing, which is now often carried out on the Illumina MiSeq and HiSeq platforms. Here, we describe a 16S rRNA gene sequencing protocol for the latest benchtop high‐throughput sequencer, the Illumina MiniSeq, which provides comparable quantity and quality of data, but at significantly reduced cost compared to larger and more expensive sequencers. This opens the opportunity for smaller labs to perform their own 16S sequencing independently.

- Next-generation sequencing (NGS) is a technology being used by many laboratories to test for inherited disorders and tumor mutations. This technology is new for many practicing pathologists, who may not be familiar with the uses, methodology, and limitations of NGS. - To familiarize pathologists with several aspects of NGS, including current and expanding uses; methodology including wet bench aspects, bioinformatics, and interpretation; validation and proficiency; limitations; and issues related to the integration of NGS data into patient care. - The review is based on peer-reviewed literature and personal experience using NGS in a clinical setting at a major academic center. - The clinical applications of NGS will increase as the technology, bioinformatics, and resources evolve to address the limitations and improve quality of results. The challenge for clinical laboratories is to ensure testing is clinically relevant, cost-effective, and can be integrated into clinical care.

• Recent studies have questioned the use of high-throughput sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region to derive a semi-quantitative representation of fungal community composition. However, comprehensive studies that quantify biases occurring during PCR and sequencing of ITS amplicons are still lacking. • We used artificially assembled communities consisting of 10 ITS-like fragments of varying lengths and guanine-cytosine (GC) contents to evaluate and quantify biases during PCR and sequencing with Illumina MiSeq, PacBio RS II and PacBio Sequel I technologies. • Fragment length variation was the main source of bias in observed community composition relative to the template, with longer fragments generally being under-represented for all sequencing platforms. This bias was three times higher for Illumina MiSeq than for PacBio RS II and Sequel I. All 10 fragments in the artificial community were recovered when sequenced with PacBio technologies, whereas the three longest fragments (> 447 bases) were lost when sequenced with Illumina MiSeq. Fragment length bias also increased linearly with increasing number of PCR cycles but could be mitigated by optimization of the PCR setup. No significant biases related to GC content were observed. • Despite lower sequencing output, PacBio sequencing was better able to reflect the community composition of the template than Illumina MiSeq sequencing.

Summary Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene‐specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site‐specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2‐edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7–100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode‐based high‐throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1‐edited T0 plants and it matched well with Sanger sequencing results. No off‐target editing was detected at the potential off‐target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.

Increased availability of soil phosphorus (P) has recently been recognised as an underlying driving factor for the positive relationship between plant diversity and ecosystem function. The effects of plant diversity on the bioavailable forms of P involved in biologically mediated rhizospheric processes and how the link between plant and soil microbial diversity facilitates soil P bioavailability, however, remain poorly understood. This study quantified four forms of bioavailable P (CaCl2‐P, citric‐P, enzyme‐P and HCl‐P) in mature subtropical forests using a novel biologically based approach, which emulates how rhizospheric processes influence the release and supply of available P. Soil microbial diversity was measured by Illumina high‐throughput sequencing. Our results suggest that tree species richness significantly affects soil microbial diversity (p < 0.05), increases litter decomposition, fine‐root biomass and length and soil organic carbon and thus increases the four forms of bioavailable P. A structural equation model that links plants, soil microbes and P forms indicated that soil bacterial and fungal diversity play dominant roles in mediating the effects of tree species richness on soil P bioavailability. An increase in the biodiversity of plants, soil bacteria and fungi could maintain soil P bioavailability and alleviate soil P limitations. Our results imply that biodiversity strengthens plant and soil feedback and increases P recycling. A plain language summary is available for this article. Plain Language Summary

Introduction The coronavirus disease (COVID‐19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), which play important roles in regulating gene expression and are also considered as essential modulators during viral infection. The aim of this study was to elucidate the differential expression of miRNAs in COVID‐19. Methods The total RNA was extracted and purified from the peripheral blood of ten patients with COVID‐19 and four healthy donors. The expression levels of various miRNAs were detected by high‐throughput sequencing, and correlation analysis was performed on the target genes that are primed by miRNAs. Key findings Compared with the healthy controls, 35 miRNAs were upregulated and 38 miRNAs were downregulated in the human patients with COVID‐19. The top 10 genes were listed below: hsa‐miR‐16‐2‐3P,hsa‐miR‐5695,hsa‐miR‐10399‐3P,hsa‐miR‐6501‐5P,hsa‐miR‐361‐3P,hsa‐miR‐361‐3p, hsa‐miR‐4659a‐3p, hsa‐miR‐142‐5p, hsa‐miR‐4685‐3p, hsa‐miR‐454‐5p, and hsa‐miR‐30c‐5p. The 10 genes with the greatest reduction were listed below: hsa‐miR‐183‐5p, hsa‐miR‐627‐5p, hsa‐miR‐941, hsa‐miR‐21‐5p, hsa‐miR‐20a‐5p, hsa‐miR‐146b‐5p, hsa‐miR‐454‐3p, hsa‐miR‐18a‐5p, hsa‐miR‐340‐5p, and hsa‐miR‐17‐5p. Remarkably, miR‐16‐2‐3p was the most upregulated miRNA, with a 1.6‐fold change compared to that of the controls. Moreover, the expression of miR‐6501‐5p and miR‐618 was 1.5‐fold higher in the COVID‐19 patients than in the healthy donors. Meanwhile, miR‐627‐5p was the most downregulated miRNA, with a 2.3‐fold change compared to that of the controls. The expression of other miRNAs (miR‐183‐5p, miR‐627‐5p, and miR‐144‐3p) was reduced by more than 1.3‐fold compared to that of the healthy donors. Cluster analysis revealed that all of the differentially expressed miRNA target genes were clustered by their regulation of cellular components, molecular functions, and biological processes. Importantly, peptidases, protein kinases, and the ubiquitin system were shown to be the highest enrichment categories by enrichment analysis. Conclusions The differential miRNA expression found in COVID‐19 patients may regulate the immune responses and viral replication during viral infection. Extract periperal blood cells from COVID‐19 patients, and micRNAs were detected by high‐throughput sequencing using illumina HiseqX Ten machine to fint the differential expression of miRNAs in COVID‐19.

Seq-Well provides similar scale and data quality to massively parallel, droplet-based single-cell RNA-seq methods in an easy to use, inexpensive and portable microwell format compatible with low-input samples. Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis .

Incidental detection of species of concern (e.g., invasive species, pathogens, threatened and endangered species) during biodiversity assessments based on high-throughput DNA sequencing holds significant risks in the absence of rigorous, fit-for-purpose data quality and reporting standards. Molecular biodiversity data are predominantly collected for ecological studies and thus are generated to common quality assurance standards. However, the detection of certain species of concern in these data would likely elicit interest from end users working in biosecurity or other surveillance contexts (e.g., pathogen detection in health-related fields), for which more stringent quality control standards are essential to ensure that data are suitable for informing decision-making and can withstand legal or political challenges. We suggest here that data quality and reporting criteria are urgently needed to enable clear identification of those studies that may be appropriately applied to surveillance contexts. In the interim, more pointed disclaimers on uncertainties associated with the detection and identification of species of concern may be warranted in published studies. This is not only to ensure the utility of molecular biodiversity data for consumers, but also to protect data generators from uncritical and potentially ill-advised application of their science in decision-making.

Forestry with short stand generations and simplified forest structures has markedly affected forest biodiversity. One group of organisms adversely affected by clear‐cutting is ectomycorrhizal (ECM) fungi, as they are associated with the roots of living trees. Retention forestry is a way of reducing logging impacts and enhancing biodiversity conservation. Increasing the proportion of trees retained at harvest may improve ECM fungal diversity. We investigated the potential for lifeboating of ECM fungi through the harvesting phase in an experimental field study in a 190‐year‐old Scots pine forest in northern Sweden. The experiment comprised four levels of tree retention—unlogged forest, plots with 60% or 30% of evenly distributed trees retained and clear‐cuts without retained trees. We sampled soils and determined identities, frequencies and relative abundances of ECM fungal species during 3 years following logging through the use of high‐throughput sequencing of amplified ITS2 markers. We identified 149 ECM fungal species, with the five most abundant species accounting for 50% of the total ECM fungal amplicons. Three years after harvesting, the proportion of ECM sequences in the total amplicon pool had decreased proportionally to the extent of tree removal. In clear‐cuts, ECM fungal relative abundance had decreased by 95%, while ECM fungal species richness had declined by 75%, compared to unlogged plots. Tree retention enabled the maintenance of the most frequent ECM species, while more lowly abundant species were progressively lost at random with increasing level of tree removal. Five of the most frequent ECM fungal species remained present after clear‐cutting, probably associated with pine seedlings. Synthesis and applications. Tree retention can moderate short‐term and potentially also long‐term logging impacts on ECM fungi. Local ECM fungal diversity is preserved in proportion to the amount of retained trees. Abundant species may be largely maintained, even by low levels of tree retention and on naturally established seedlings. However, conservation of more infrequent species requires higher levels of tree retention, and our results suggest that around 75% of the ECM species are lost with the forest certification standard of 5% retention trees left at logging. Tree retention can moderate short‐term and potentially also long‐term logging impacts on ECM fungi. Local ECM fungal diversity is preserved in proportion to the amount of retained trees. Abundant species may be largely maintained, even by low levels of tree retention and on naturally established seedlings. However, conservation of more infrequent species requires higher levels of tree retention, and our results suggest that around 75% of the ECM species are lost with the forest certification standard of 5% retention trees left at logging.