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Despite many efforts to understand and leverage the functional potential of environmental viromes, most bacteriophage genes are largely uncharacterized. To explore novel biology from uncultivated microbes like phages, metagenomics has emerged as a powerful tool to directly mine new genes without the need to culture the diverse microbiota and the viruses within. When a pure computational approach cannot infer gene function, it may be necessary to create a DNA library from environmental genomic DNA, followed by the screening of that library for a particular function. However, these screens are often initiated without a metagenomic analysis of the completed DNA library being reported. Here, we describe the construction and characterization of DNA libraries from a single cultured phage (FT4), five cultured Escherichia coli phages, and three metagenomic viral sets built from freshwater, seawater, and wastewater samples. Through next-generation sequencing of five independent samplings of the libraries, we found a consistent number of recovered genes per replicate for each library, with many genes classifiable via the KEGG and Pharokka databases. By characterizing the size of the genes and inserts, we found that our libraries contain a median of one to two genes per contig with a median gene length of 303-381 bp for all libraries, reflective of the small genomes of viruses. The environmental libraries were genetically diverse compared to the single phage and multi-phage libraries. Additionally, we found reduced coverage of individual genomes when five phages were used as opposed to one. Taken together, this work provides a comprehensive analysis of the DNA libraries from phage genomes that can be used for metagenomic exploration and functional screens to infer and identify new biology. IMPORTANCE Functional metagenomics is an approach that aims to characterize the putative biological function of genes in the microbial world. This includes an examination of the sequencing data collected from a pooled source of diverse microbes and inference of gene function by comparison to annotated and studied genes from public databases. At times, DNA libraries are made from these genes, and the library is screened for a specific function. Hits are validated using a combination of biological, computational, and structural analysis. Left unresolved is a detailed characterization of the library, both its diversity and content, for the purposes of imputing function entirely by computational means, a process that may yield findings that aid in designing useful screens to identify novel gene functions. In this study, we constructed libraries from cultured phages and uncultured viromes from the environment and characterized some important parameters, such as gene number, genes per contig, ratio of hypothetical to known proteins, total genomic coverage and recovery, and the effect of pooling genetic information from multiple sources, to provide a better understanding of the nature of these libraries. This work will aid the design and implementation of future screens of pooled DNA libraries to discover and isolate viral genes with novel biology across various biomes. [ABSTRACT FROM AUTHOR]

Abstract The low costs of array‐synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost‐effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site‐specific recombination to index library DNA, and next‐generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost‐effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.

The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. A learning-by-building approach can now answer questions about how chromosomes must be constructed to maintain genetic information. Here we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular modular cloning (MoClo) system., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Disrupted circadian rhythms is a prominent and early feature of neurodegenerative diseases including Huntington’s disease (HD). In HD patients and animal models, striatal and hypothalamic neurons expressing molecular circadian clocks are targets of mutant Huntingtin (mHtt) pathogenicity. Yet how mHtt disrupts circadian rhythms remains unclear. In a genetic screen for modifiers of mHtt effects on circadian behavior in Drosophila, we discovered a role for the neurodegenerative disease gene Ataxin2 (Atx2). Genetic manipulations of Atx2 modify the impact of mHtt on circadian behavior as well as mHtt aggregation and demonstrate a role for Atx2 in promoting mHtt aggregation as well as mHtt-mediated neuronal dysfunction. RNAi knockdown of the Fragile X mental retardation gene, dfmr1, an Atx2 partner, also partially suppresses mHtt effects and Atx2 effects depend on dfmr1. Atx2 knockdown reduces the cAMP response binding protein A (CrebA) transcript at dawn. CrebA transcript level shows a prominent diurnal regulation in clock neurons. Loss of CrebA also partially suppresses mHtt effects on behavior and cell loss and restoration of CrebA can suppress Atx2 effects. Our results indicate a prominent role of Atx2 in mediating mHtt pathology, specifically via its regulation of CrebA, defining a novel molecular pathway in HD pathogenesis. [ABSTRACT FROM AUTHOR]

Interception of potential invasive species at ports-of-entry is essential for effective biosecurity and biosurveillance programs. However, taxonomic assessment of the immature stages of most arthropods is challenging; characters for identification are often dependent on adult morphology and reproductive structures. This study aims to strengthen the identification of such specimens through DNA barcoding, with a focus on microlepidoptera. A sample of 241 primarily immature microlepidoptera specimens intercepted at U.S. ports-of-entry from 2007 to 2011 were selected for analysis. From this sample, 201 COI-5P sequences were generated and analyzed for concordance between morphology-based and DNA-based identifications. The retrospective analysis of the data over 10 years (2009 to 2019) using the Barcode of Life Data (BOLD) system demonstrates the importance of establishing and growing DNA barcode reference libraries for use in specimen identification. Additionally, analysis of specimen identification using public data (43.3% specimens identified) vs. non-public data (78.6% specimens identified) highlights the need to encourage researchers to make data publicly accessible. DNA barcoding surpassed morphological identification with 42.3% (public) and 66.7% (non-public) of the sampled specimens achieving a species-level identification, compared to 38.3% species-level identification by morphology. Whilst DNA barcoding was not able to identify all specimens in our dataset, its incorporation into border security programs as an adjunct to morphological identification can provide secondary lines of evidence and lower taxonomic resolution in many cases. Furthermore, with increased globalization, database records need to be clearly annotated for suspected specimen origin versus interception location. [ABSTRACT FROM AUTHOR]

Globally, crustaceans represent one of the most taxonomically diverse and economically important invertebrate group. Notwithstanding, the diversity within this group is poorly known because most crustaceans are often associated with varied habits, forms, sizes and habitats; making species identification by conventional methods extremely challenging. In addition, progress towards understanding the diversity within this group especially in southern Africa has been severely hampered by the declining number of trained taxonomists, the presence of invasive alien species, over exploitation, etc. However, the advent of molecular techniques such as “DNA barcoding and Metabarcoding” can accelerate species identification and the discovery of new species. To contribute to the growing body of knowledge on crustacean diversity, we collected data from five southern African countries and used a DNA barcoding approach to build the first DNA barcode reference library for southern African crustaceans. We tested the reliability of this DNA barcode reference library to facilitate species identification using two approaches. We recovered high efficacy of specimen identification/discrimination; supported by both barcode gap and tree-base species identification methods. In addition, we identified alien invasive species and specimens with ‘no ID” in our DNA barcode reference library. The later; highlighting specimens requiring (i) further investigation and/or (ii) the potential presence of cryptic diversity or (iii) misidentifications. This unique data set although with some sampling gaps presents many opportunities for exploring the effect and extent of invasive alien species, the role of the pet trade as a pathway for crustacean species introduction into novel environments, sea food authentication, phylogenetic relationships within the larger crustacean groupings and the discovery of new species. [ABSTRACT FROM AUTHOR]

In this study, we describe the development of a PCR-free whole exome sequencing method. Using this method, 2 μg DNA was sufficient for library preparation for whole exome sequencing. Furthermore, the method is simple and makes use of a commercial kit, with additional step of concentrating the captured library by ethanol precipitation. The accuracy of the PCR-free method was found to be equivalent to that of unique molecular identifier-corrected analysis method, which is the commonly used method to detect rare mutations. Thus, the PCR-free whole exome sequencing method is cost-effective as well as efficient in detecting rare mutations. [ABSTRACT FROM AUTHOR]

CD36 is a platelet membrane glycoprotein whose engagement with oxidized low-density lipoprotein (oxLDL) results in platelet activation. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk by genome-wide association studies. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative. We screened 81 expression quantitative trait loci (eQTL) single nucleotide polymorphisms (SNPs) associated with platelet CD36 expression by a Massively Parallel Reporter Assay (MPRA) and analyzed the results with a novel Bayesian statistical method. Ten eQTLs located 13kb to 55kb upstream of the CD36 transcriptional start site of transcript ENST00000309881 and 49kb to 92kb upstream of transcript ENST00000447544, demonstrated significant transcription shifts between their minor and major allele in the MPRA assay. Of these, rs2366739 and rs1194196, separated by only 20bp, were confirmed by luciferase assay to alter transcriptional regulation. In addition, electromobility shift assays demonstrated differential DNA:protein complex formation between the two alleles of this locus. Furthermore, deletion of the genomic locus by CRISPR/Cas9 in K562 and Meg-01 cells results in upregulation of CD36 transcription. These data indicate that we have identified a variant that regulates expression of CD36, which in turn affects platelet function. To assess the clinical relevance of our findings we used the PhenoScanner tool, which aggregates large scale GWAS findings; the results reinforce the clinical relevance of our variants and the utility of the MPRA assay. The study demonstrates a generalizable paradigm for functional testing of genetic variants to inform mechanistic studies, support patient management and develop precision therapies. [ABSTRACT FROM AUTHOR]

Alopecia areata (AA) is a hair loss disorder resulting from an autoimmune reaction against hair follicles. T-helper 1 cells are a major contributor to this disorder, but little is known about the role of T-regulatory cells (Tregs) in AA. Here, we analysed the distribution of circulating Treg subsets in twenty AA patients with active hair loss and fifteen healthy subjects by flow cytometry. The Treg suppressor HLA-DR+ subpopulation was significantly reduced in the patients (P<0.001) and there were significantly fewer cells expressing CD39 among the CD4+CD25+Foxp3+ Treg subpopulation in patients (P = 0.001). FOXP3 CD39 Treg cells were also reduced in hair follicles; by 75% in non-lesional skin and 90% in lesional skin, when compared to control healthy skin. To further characterise Treg cells in AA; Tregs (CD4+CD25+FOXP3+) were investigated for their TCRβ sequence. PCR products analysed by Next Generation Sequencing techniques, showed that all frequent public clonotypes in AA Tregs were also present in controls at relatively similar frequencies, excepting two public clonotypes: (V15 D1 J1-4) and (V2 D1 J1-2), which were exclusively present in controls. This suggests that these Treg clonotypes may have a protective effect and that they may be an exciting subject for future therapeutic applications. [ABSTRACT FROM AUTHOR]

Kidney failure occurs in 5–13% of individuals with sickle cell disease and is associated with early mortality. Two APOL1 alleles (G1 and G2) have been identified as risk factors for sickle cell disease nephropathy. Both risk alleles are prevalent in individuals with recent African ancestry and have been associated with nephropathic complications in other diseases. Despite the association of G1 and G2 with kidney dysfunction, the mechanisms by which these variants contribute to increased risk remain poorly understood. Previous work in zebrafish models suggest that the G2 risk allele functions as a dominant negative, whereas the G1 allele is a functional null. To understand better the cellular pathology attributed to APOL1 G2, we investigated the in vivo effects of the G2 risk variant on distinct cell types using RNA sequencing. We surveyed APOL1 G2 associated transcriptomic alterations in podocytes and vascular endothelial cells isolated from zebrafish larvae expressing cell-type specific reporters. Our analysis identified many transcripts (n = 7,523) showing differential expression between APOL1 G0 (human wild-type) and APOL1 G2 exposed podocytes. Conversely, relatively few transcripts (n = 107) were differentially expressed when comparing APOL1 G0 and APOL1 G2 exposed endothelial cells. Pathway analysis of differentially expressed transcripts in podocytes showed enrichment for autophagy associated terms such as “Lysosome” and “Phagosome”, implicating these pathways in APOL1 G2 associated kidney dysfunction. This work provides insight into the molecular pathology of APOL1 G2 nephropathy which may offer new therapeutic strategies for multiple disease contexts such as sickle cell nephropathy. [ABSTRACT FROM AUTHOR]

Development of a robust automated platform for enrichment of extracellular vesicles from low sample volume that matches the needs for next-generation sequencing could remove major hurdles for genomic biomarker discovery. Here, we document a protocol for urinary EVs enrichment by utilizing an automated microfluidic system, termed acoustic trap, followed by next-generation sequencing of microRNAs (miRNAs) for biomarker discovery. Specifically, we compared the sequencing output from two small RNA library preparations, NEXTFlex and CATS, using only 130 pg of input total RNA. The samples prepared using NEXTflex was found to contain larger number of unique miRNAs that was the predominant RNA species whereas rRNA was the dominant RNA species in CATS prepared samples. A strong correlation was found between the miRNA expressions of the acoustic trap technical replicate in the NEXTFlex prepared samples, as well as between the acoustic trap and ultracentrifugation enrichment methods. Together, these results demonstrate a robust and automated strategy for biomarker discovery from small volumes of urine. [ABSTRACT FROM AUTHOR]

Morphological study of 1,795 spiders from sites across Pakistan placed these specimens in 27 families and 202 putative species. COI sequences >400 bp recovered from 1,782 specimens were analyzed using neighbor-joining trees, Bayesian inference, barcode gap, and Barcode Index Numbers (BINs). Specimens of 109 morphological species were assigned to 123 BINs with ten species showing BIN splits, while 93 interim species included representatives of 98 BINs. Maximum conspecific divergences ranged from 0–5.3% while congeneric distances varied from 2.8–23.2%. Excepting one species pair (Oxyopes azhari–Oxyopes oryzae), the maximum intraspecific distance was always less than the nearest-neighbor (NN) distance. Intraspecific divergence values were not significantly correlated with geographic distance. Most (75%) BINs detected in this study were new to science, while those shared with other nations mainly derived from India. The discovery of many new, potentially endemic species and the low level of BIN overlap with other nations highlight the importance of constructing regional DNA barcode reference libraries. [ABSTRACT FROM AUTHOR]

Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus. [ABSTRACT FROM AUTHOR]

Formalin-fixed paraffin-embedded (FFPE) tissues are among the most widely available clinical specimens. Their potential utility as a source of RNA for transcriptome studies would greatly enhance population-based cancer studies. Although preliminary studies suggest FFPE tissue may be used for RNA sequencing, the effect of storage time on these specimens needs to be determined. We conducted this study to determine whether RNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries was present in sufficient quantity and quality for RNA-Seq analysis. FFPE tissues, stored from 7 to 32 years, were obtained from three SEER sites. RNA was extracted, quantified, quality assessed, and subjected to RNA-Seq (a whole transcriptome sequencing technology). FFPE specimens stored for longer periods of time had poorer RNA sample quality as indicated by negative correlations between specimen storage time and fragment distribution values (DV). In addition, sample contamination was a common issue among the RNA, with 41 of 67 samples having 5% to 48% bacterial contamination. However, regardless of specimen storage time and bacterial contamination, 60% of the samples yielded data that enabled gene expression quantification, identifying more than 10,000 genes, with the correlations among most biological replicates above 0.7. This study demonstrates that FFPE high-grade ovarian serous adenocarcinomas specimens stored in repositories for up to 32 years and under varying storage conditions are a promising source of RNA for RNA-Seq. We also describe certain caveats to be considered when designing RNA-Seq studies using archived FFPE tissues. [ABSTRACT FROM AUTHOR]

Abstract This work presents an algorithm for constructing artificial DNA libraries that consist of chains having minimal tendency to hybridize with each other. Such libraries can be applied in DNA computing (Adleman, 1994) for encoding problem instances. The algorithm is based on an extension of de Bruijn graphs and de Bruijn sequences. In general, the algorithm can be used to build a chain over any alphabet of even size with defined binary symmetric irreflexive relation of complementarity — the obtained chain is a modified de Bruijn sequence with shifted windows (there are no repetitive k -long subchains that start on positions divisible by a given parameter) where half of these windows are removed to eliminate relation of complementarity among them. [ABSTRACT FROM AUTHOR]

Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in next generation sequencing (NGS) is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE samples of varying tissue types. Quality assessment included total yield, percent dsDNA, fragment analysis and multiplex PCR. After assessment, three tissue types from four FFPE DNA methods were selected for NGS downstream evaluation, targeted and whole exome sequencing. In addition, two low input library protocols were evaluated for WES. Analysis revealed average coverage across the target regions for WES was ~20-30X for all four FFPE DNA extraction methods. For the targeted panels, the highest molecular tag coverage was obtained with the Kingfisher FFPE extraction method. The genotype concordance was 99% for the commonly called variant positions between all four extraction methods with the targeted PCR NGS panel and 96% with WES. Assessing quality of extracted DNA aids in selecting the optimal NGS approach, and the choice of both DNA extraction and library preparation approaches can impact the performance of archival tissue in NGS. [ABSTRACT FROM AUTHOR]

Fragmentation of DNA is the very important first step in preparing nucleic acids for next-generation sequencing. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is a simple, robust, and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for direct use in NGS library construction and allows the elimination of the additional step of reparation of DNA ends. [ABSTRACT FROM AUTHOR]

A fundamental assumption, common to the vast majority of high-throughput transcriptome analyses, is that the expression of most genes is unchanged among samples and that total cellular RNA remains constant. As the number of analyzed experimental systems increases however, different independent studies demonstrate that this assumption is often violated. We present a calibration method using RNA spike-ins that allows for the measurement of absolute cellular abundance of RNA molecules. We apply the method to pooled RNA from cell populations of known sizes. For each transcript, we compute a nominal abundance that can be converted to absolute by dividing by a scale factor determined in separate experiments: the yield coefficient of the transcript relative to that of a reference spike-in measured with the same protocol. The method is derived by maximum likelihood theory in the context is a complete statistical model for sequencing counts contributed by cellular RNA and spike-ins. The counts are based on a sample from a fixed number of cells to which a fixed population of spike-in molecules has been added. We illustrate and evaluate the method with applications to two global expression data sets, one from the model eukaryote Saccharomyces cerevisiae, proliferating at different growth rates, and differentiating cardiopharyngeal cell lineages in the chordate Ciona robusta. We tested the method in a technical replicate dilution study, and in a k-fold validation study. [ABSTRACT FROM AUTHOR]

Background: KRAS and NRAS mutations are identified resistance mutations to anti-epidermal growth factor receptor monoclonal antibodies in patients with metastatic colorectal cancer. BRAF status is also routinely assessed for its poor prognosis value. In our institute, next-generation sequencing (NGS) is routinely used for gene-panel mutations detection including KRAS, NRAS and BRAF, but DNA quality is sometimes not sufficient for sequencing. In our routine practice, Idylla platform is used for the analysis of samples that don’t reach sufficient quality criteria for NGS assay. Methods: In this study, data from mCRC samples analyzed from May 2017 to 2018 were retrospectively collected. All samples with a poor DNA quality for sequencing have been assessed using Idylla platform. First, KRAS Idylla assay cartridge has been used for the determination of KRAS mutational status. All KRAS wild-type samples have then been analyzed using NRAS-BRAF assay. Among 669 samples, 67 samples failed the DNA quality control and have been assessed on Idylla KRAS mutation test. Results: Among 67 samples, 50 (75%) samples had a valid result with Idylla KRAS mutation test including 22 carrying a KRAS mutation. For 28 samples, NRAS and BRAF mutational statuses have been assessed using Idylla NRAS-BRAF mutation test. Among 28 samples, 27 (96%) had a valid result including 2 samples bearing a NRAS mutation and 3 samples bearing a BRAF mutation. Conclusions: Our study shows that an integrated workflow using NGS and Idylla platform allows the determination of KRAS, NRAS and BRAF mutational statuses of 651/669 (97.3%) samples and retrieve 49/67 (73.1%)samples that don’t reach DNA quality requirements for NGS. [ABSTRACT FROM AUTHOR]

Selection of aptamers from oligonucleotide libraries currently requires multiple rounds of alternating steps of partitioning of binders from nonbinders and enzymatic amplification of all collected oligonucleotides. Herein, we report a highly practical solution for reliable one‐step selection of aptamers. We introduce partitioning by ideal‐filter capillary electrophoresis (IFCE) in which binders and nonbinders move in the opposite directions. The efficiency of IFCE‐based partitioning reaches 109, which is ten million times higher than that of typical solid‐phase partitioning methods. One step of IFCE‐based partitioning is sufficient for the selection of a high‐affinity aptamer pool for a protein target. Partitioning by IFCE promises to become an indispensable tool for fast and robust selection of binders from different types of oligonucleotide libraries. A good selection: Ideal‐filter capillary electrophoresis (IFCE) is introduced to facilitate the reliable and practical one‐step selection of oligonucleotide aptamers for protein targets. IFCE will also be applicable to the selection of binders from DNA‐encoded libraries of small molecules. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) requires RNA-dependent RNA polymerases (RdRPs) in many eukaryotes, and RNAi amplification constitutes the only known function for eukaryotic RdRPs. Yet in animals, classical model organisms can elicit RNAi without possessing RdRPs, and only nematode RNAi was shown to require RdRPs. Here we show that RdRP genes are much more common in animals than previously thought, even in insects, where they had been assumed not to exist. RdRP genes were present in the ancestors of numerous clades, and they were subsequently lost at a high frequency. In order to probe the function of RdRPs in a deuterostome (the cephalochordate Branchiostoma lanceolatum), we performed high-throughput analyses of small RNAs from various Branchiostoma developmental stages. Our results show that Branchiostoma RdRPs do not appear to participate in RNAi: we did not detect any candidate small RNA population exhibiting classical siRNA length or sequence features. Our results show that RdRPs have been independently lost in dozens of animal clades, and even in a clade where they have been conserved (cephalochordates) their function in RNAi amplification is not preserved. Such a dramatic functional variability reveals an unexpected plasticity in RNA silencing pathways. [ABSTRACT FROM AUTHOR]

Retrotransposon expression during arbuscular mycorrhizal (AM) fungal colonisation of sunflower roots (Helianthus annuus) was analysed using Illumina RNA-Seq, in order to verify whether mycorrhizal symbiosis can activate retrotransposable elements. Illumina cDNA libraries were produced from RNAs isolated from the roots of sunflower plants at 4 and 16 days after inoculation with the AM fungus Rhizoglomus irregulare and from their respective control plants. Illumina reads were mapped to a library of reverse transcriptase-encoding sequences, putatively belonging to long terminal repeat retrotransposons of Gypsy and Copia superfamilies. Forty-six different reverse transcriptase sequences were transcribed, although at a low rate, in mycorrhizal or control roots and only four were significantly over-expressed at day 16, compared with control roots. Almost all expressed or over-expressed sequences belonged to low-copy elements, mostly, of the Copia superfamily. A meta-analysis, using publicly available Illumina cDNA libraries obtained from sunflower plants treated with different hormones and chemicals, mimicking stimuli produced by abiotic and biotic stresses, was also conducted. Such analyses indicated that the four reverse transcriptase sequences over-expressed in mycorrhizal roots were explicitly induced only by AM symbiosis, showing the specificity of AM stimuli compared to that of other fungal/plant interactions. [ABSTRACT FROM AUTHOR]

Current progress in the field of next-generation transcriptome sequencing have contributed significantly to the study of various malignancies including glioblastoma multiforme (GBM). Differential sequencing of transcriptomes of patients and non-tumor controls has a potential to reveal novel transcripts with significant role in GBM. One such candidate group of molecules are long non-coding RNAs (lncRNAs) which have been proved to be involved in processes such as carcinogenesis, epigenetic modifications and resistance to various therapeutic approaches. To maximize the value of transcriptome sequencing, a proper protocol for library preparation from tissue-derived RNA needs to be found which would produce high quality transcriptome sequencing data and increase the number of detected lncRNAs. It is important to mention that success of library preparation is determined by the quality of input RNA, which is in case of real-life tissue specimens very often altered in comparison to high quality RNA commonly used by manufacturers for development of library preparation chemistry. In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Libraries generated using SENSE kit were characterized by the most normal distribution of normalized average GC content, the least amount of over-represented sequences and the percentage of ribosomal RNA reads (0.3–1.5%) and highest numbers of uniquely mapped reads and reads aligning to coding regions. However, NEBNext kit performed better having relatively low duplication rates, even transcript coverage and the highest number of hits in Ensembl database for every biotype of our interest including lncRNAs. Our results indicate that out of three approaches the NEBNext library preparation kit was most suitable for the study of lncRNAs via transcriptome sequencing. This was further confirmed by highly consistent data reached in an independent validation on an expanded cohort. [ABSTRACT FROM AUTHOR]

Hybridization capture with in-solution oligonucleotide probes has quickly become the preferred method for enriching specific DNA loci from degraded or ancient samples prior to high-throughput sequencing (HTS). Several companies synthesize sets of probes for in-solution hybridization capture, but these commercial reagents are usually expensive. Methods for economical in-house probe synthesis have been described, but they do not directly address one of the major advantages of commercially synthesised probes: that probe sequences matching many species can be synthesised in parallel and pooled. The ability to make “phylogenetically diverse” probes increases the cost-effectiveness of commercial probe sets, as they can be used across multiple projects (or for projects involving multiple species). However, it is labour-intensive to replicate this with in-house methods, as template molecules must first be generated for each species of interest. While it has been observed that probes can be used to enrich for phylogenetically distant targets, the ability of this effect to compensate for the lack of phylogenetically diverse probes in in-house synthesised probe sets has not been tested. In this study, we present a refined protocol for in-house RNA probe synthesis and evaluated the ability of probes generated using this method from a single species to successfully enrich for the target locus in phylogenetically distant species. We demonstrated that probes synthesized using long-range PCR products from a placental mammal mitochondrion (Bison spp.) could be used to enrich for mitochondrial DNA in birds and marsupials (but not plants). Importantly, our results were obtained for approximately a third of the cost of similar commercially available reagents. [ABSTRACT FROM AUTHOR]

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved cost savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library. [ABSTRACT FROM AUTHOR]