Metabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ...ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80–90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.
Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed ...a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA).
An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping.
NGS could identify mutations present in DNA dilutions at ≥ 0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification.
Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research.
Mammalian microRNAs (miRNAs) pair to 3′UTRs of mRNAs to direct their posttranscriptional repression. Important for target recognition are ∼7 nt sites that match the seed region of the miRNA. However, ...these seed matches are not always sufficient for repression, indicating that other characteristics help specify targeting. By combining computational and experimental approaches, we uncovered five general features of site context that boost site efficacy: AU-rich nucleotide composition near the site, proximity to sites for coexpressed miRNAs (which leads to cooperative action), proximity to residues pairing to miRNA nucleotides 13–16, positioning within the 3′UTR at least 15 nt from the stop codon, and positioning away from the center of long UTRs. A model combining these context determinants quantitatively predicts site performance both for exogenously added miRNAs and for endogenous miRNA-message interactions. Because it predicts site efficacy without recourse to evolutionary conservation, the model also identifies effective nonconserved sites and siRNA off-targets.
Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress. Our previous study demonstrated that disturbed flow with low and oscillatory shear stress (OSS) induces ...bone morphogenetic protein receptor (BMPR)-specific Smad1/5 activation in ECs, but the underlying mechanisms and the in vivo functional role of Smad1/5 remain unclear.
Here we elucidated the molecular mechanisms by which OSS activates EC Smad1/5 and its in vivo functional role.
Lentiviral Smad5-specific short hairpin RNA (Lenti-shSmad5) was constructed and intra-arterially injected into the lumen of stenosed abdominal aorta in bromodeoxyuridine-infused rats. Co-immunoprecipitation and in situ proximity ligation assays were performed on ECs exposed to OSS (0.5 ± 4 dynes/cm(2) ) in a parallel-plate flow chamber to investigate BMPR-integrin interactions and their regulatory role in OSS-activation of EC Smad1/5.
Intra-arterial administration of Lenti-shSmad5 inhibited bromodeoxyuridine uptake of ECs at post-stenotic sites, where disturbed flow with OSS occurs. OSS induced sustained BMPRIB-αv β3 integrin association in ECs, which was mediated by the intracytoplasmic kinase domain of BMPRII and subsequently activated the Shc/focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) cascade, leading to Smad1/5 activation. This OSS-activation of Smad1/5 induced its association with and activation of runt-related transcription factor-2 (Runx2), leading to activations of mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K), a pathway critical for EC proliferation in response to OSS.
Oscillatory shear stress induces synergistic interactions between specific BMPRs and integrin to activate Smad1/5 through the Shc/FAK/ERK pathway, which leads to the activation of the Runx2/mTOR/p70S6K pathway to promote EC proliferation.
Thousands of mammalian messenger RNAs are under selective pressure to maintain 7-nucleotide sites matching microRNAs (miRNAs). We found that these conserved targets are often highly expressed at ...developmental stages before miRNA expression and that their levels tend to fall as the miRNA that targets them begins to accumulate. Nonconserved sites, which outnumber the conserved sites 10 to 1, also mediate repression. As a consequence, genes preferentially expressed at the same time and place as a miRNA have evolved to selectively avoid sites matching the miRNA. This phenomenon of selective avoidance extends to thousands of genes and enables spatial and temporal specificities of miRNAs to be revealed by finding tissues and developmental stages in which messages with corresponding sites are expressed at lower levels.
•Next-generation sequencing (NGS) of cell-free DNA could expand access to precision medicine.•Technical differences between plasma NGS assays are poorly characterized.•We compare complex variants ...detection between two plasma NGS assays.•Using tumor as a reference, we identify biochemical and bioinformatic differences.
Plasma genotyping represents an opportunity for convenient detection of clinically actionable mutations in advanced cancer patients, such has been well-documented in non-small cell lung cancer (NSCLC). Oncogenic gene fusions are complex variants that may be more challenging to detect by next-generation sequencing (NGS) of plasma cell-free DNA (cfDNA). Rigorous evaluation of plasma NGS assays in the detection of fusions is needed to maximize clinical utility.
: Additional plasma was collected from patients with advanced NSCLC and ALK, ROS1, or RET gene fusions in tissue who had undergone clinical plasma NGS using Guardant360™(G360, Guardant Health). We then sequenced extracted cfDNA with a plasma NGS kit focused on known driver mutations in NSCLC (ctDx-Lung, Resolution Bioscience) with cloud-based bioinformatic analysis and blinded variant calling.
Of 16 patients assayed known to harbor anALK, ROS1, or RET in tumor, G360 detected fusions in 7 cases, ctDx-Lung detected fusions in 13 cases, and 3 cases were detected by neither. Of the 7 fusions detected by both assays, G360 reported lower mutant allelic fractions (AF). In cases missed by G360, tumor derived TP53 mutations were often detected confirming presence of tumor DNA. Raw sequencing data showed that inverted or out-of-frame variants were overrepresented in cases detected using ctDx-Lung but not by G360.
Focusing on complex, clinically actionable mutations using tumor as a reference standard allows for evaluation of technical differences in plasma NGS assays that may impact clinical performance. Noting the heterogeneity of fusion sequences observed in NSCLC, we hypothesize that differences in hybrid capture techniques and bioinformatic calling may be sources of variations in sensitivity among these assays.
Chinese hamster ovary (CHO) cells are the most prevalent mammalian cell factories for producing recombinant therapeutic proteins due to their ability to synthesize human‐like post‐translational ...modifications and ease of maintenance in suspension cultures. Currently, a wide variety of CHO host cell lines has been developed; substantial differences exist in their phenotypes even when transfected with the same target vector. However, relatively less is known about the influence of their inherited genetic heterogeneity on phenotypic traits and production potential from the bioprocessing point of view. Herein, we present a global transcriptome and proteome profiling of three commonly used parental cell lines (CHO‐K1, CHO‐DXB11, and CHO‐DG44) in suspension cultures and further report their growth‐related characteristics, and N‐ and O‐glycosylation patterns of host cell proteins (HCPs). The comparative multi‐omics and subsequent genome‐scale metabolic network model‐based enrichment analyses indicated that some physiological variations of CHO cells grown in the same media are possibly originated from the genetic deficits, particularly in the cell‐cycle progression. Moreover, the dihydrofolate reductase deficient DG44 and DXB11 possess relatively less active metabolism when compared to K1 cells. The protein processing abilities and the N‐ and O‐glycosylation profiles also differ significantly across the host cell lines, suggesting the need to select host cells in a rational manner for the cell line development on the basis of recombinant protein being produced.
A mammalian systems biotechnology approach exploited multi‐omics profiles together with genome‐scale metabolic network model to systematically link the possible phenotypic differences across Chinese hamster ovary (CHO) host cell lines with their genotypes. The current work highlights that the genetic diversity and heterogeneity of CHO cells indeed affect their growth phenotype as well as glycosylation patterns, thus serving as a good starting point for the rational selection of relevant CHO host cells as well as to establish cell line engineering targets.
Trade in pangolins is illegal, and yet tons of their scales and products are seized at various ports. These large seizures are challenging to process and comprehensively genotype for upstream ...provenance tracing and species identification for prosecution. We implemented a scalable DNA barcoding pipeline in which rapid DNA extraction and MinION sequencing were used to genotype a substantial proportion of pangolin scales subsampled from 2 record shipments seized in Singapore in 2019 (37.5 t). We used reference sequences to match the scales to phylogeographical regions of origin. In total, we identified 2346 cytochrome b (cytb) barcodes of white‐bellied (Phataginus tricuspis) (from 1091 scales), black‐bellied (Phataginus tetradactyla) (227 scales), and giant (Smutsia gigantea) (1028 scales) pangolins. Haplotype diversity was higher for P. tricuspis scales (121 haplotypes, 66 novel) than that for P. tetradactyla (22 haplotypes, 15 novel) and S. gigantea (25 haplotypes, 21 novel) scales. Of the novel haplotypes, 74.2% were likely from western and west‐central Africa, suggesting potential resurgence of poaching and newly exploited populations in these regions. Our results illustrate the utility of extensively subsampling large seizures and outline an efficient molecular approach for rapid genetic screening that should be accessible to most forensic laboratories and enforcement agencies.
Revelación de la magnitud de la caza furtiva del pangolín africano mediante el genotipo extenso de nanoporos de ADN de escamas incautadas
Resumen
Aunque el mercado de pangolines es ilegal, se incautan toneladas de sus escamas y productos derivados en varios puertos comerciales. Es un reto procesar estas magnas incautaciones y obtener el genotipo completo para usarlo en la trazabilidad logística ascendente e identificación de la especie y así imponer sanciones. Implementamos una canalización escalable del código de barras de ADN en el cual usamos la extracción rápida de ADN y la secuenciación MinION para obtener el genotipo de una proporción sustancial de las escamas de pangolín submuestreadas en dos cargamentos incautados en 2019 en Singapur (37.5 t). Usamos secuencias referenciales para emparejar las escamas con las regiones filogeográficas de origen. Identificamos en total 2,346 códigos de citocromo b (cytb) del pangolín de vientre blanco (Phataginus tricuspis) (de 1,091 escamas), de vientre negro (P. tetradactyla) (227 escamas) y del pangolín gigante (Smutsia gigantea) (1,028 escamas). La diversidad de haplotipos fue mayor en las escamas de P. tricuspis (121 haplotipos, 66 nuevos) que en las de P. tetradactyla (22 haplotipos, 15 nuevos) y S. gigantea (25 haplotipos, 21 nuevos). De los haplotipos nuevos, el 74.2% probablemente provenía del occidente y centro‐occidente de África, lo que sugiere un resurgimiento potencial de la caza furtiva y poblaciones recién explotadas en estas regiones. Nuestros resultados demuestran la utilidad de submuestrear extensivamente las grandes incautaciones y esboza una estrategia molecular eficiente para un análisis genético rápido que debería ser accesible para la mayoría de los laboratorios forenses y las autoridades de aplicación.
Rapid diagnosis is one key pillar to end tuberculosis (TB). Point-of-care tests (POCTs) facilitate early detection, immediate treatment, and reduced transmission of TB disease. This Review evaluates ...current diagnostic assays endorsed by the World Health Organization and identifies the gaps between existing conventional tests and the ideal POCT. We discuss the commercial development of new rapid tests and research studies on nonsputum-based diagnostic biomarkers from both pathogen and host. Last, we highlight advances in integrated microfluidics technology that may aid the development of new POCTs.
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