The large collections of Arabidopsis thaliana sequence-indexed T-DNA insertion mutants are among the most important resources to emerge from the sequencing of the genome. Several laboratories around ...the world have used the Arabidopsis reference genome sequence to map T-DNA flanking sequence tags (FST) for over 325 000 T-DNA insertion lines. Over the past decade, phenotypes identified with T-DNA-induced mutants have played a critical role in advancing both basic and applied plant research. These widely used mutants are an invaluable tool for direct interrogation of gene function. However, most lines are hemizygous for the insertion, necessitating a genotyping step to identify homozygous plants for the quantification of phenotypes. This situation has limited the application of these collections for genome-wide screens. Isolating multiple homozygous insert lines for every gene in the genome would make it possible to systematically test the phenotypic consequence of gene loss under a wide variety of conditions. One major obstacle to achieving this goal is that 12% of genes have no insertion and 8% are only represented by a single allele. Generation of additional mutations to achieve full genome coverage has been slow and expensive since each insertion is sequenced one at a time. Recent advances in high-throughput sequencing technology open up a potentially faster and cost-effective means to create new, very large insertion mutant populations for plants or animals. With the combination of new tools for genome-wide studies and emerging phenotyping platforms, these sequence-indexed mutant collections are poised to have a larger impact on our understanding of gene function.
Advances in the biological sciences have led to an ongoing paradigm shift in toxicity testing based on expanded application of high-throughput in vitro screening and in silico methods to assess ...potential health risks of environmental agents. This review examines progress on the vision for toxicity testing elaborated by the US National Research Council (NRC) during the decade that has passed since the 2007 NRC report on
Toxicity Testing in the 21st Century
(TT21C). Concomitant advances in exposure assessment, including computational approaches and high-throughput exposomics, are also documented. A vision for the next generation of risk science, incorporating risk assessment methodologies suitable for the analysis of new toxicological and exposure data, resulting in human exposure guidelines is described. Case study prototypes indicating how these new approaches to toxicity testing, exposure measurement, and risk assessment are beginning to be applied in practice are presented. Overall, progress on the 20-year transition plan laid out by the US NRC in 2007 has been substantial. Importantly, government agencies within the United States and internationally are beginning to incorporate the new approach methodologies envisaged in the original TT21C vision into regulatory practice. Future perspectives on the continued evolution of toxicity testing to strengthen regulatory risk assessment are provided.
Summary
Monoclonal antibody discovery and engineering is a field that has traditionally been dominated by high‐throughput screening platforms (e.g. hybridomas and surface display). In recent years ...the emergence of high‐throughput sequencing has made it possible to obtain large‐scale information on antibody repertoire diversity. Additionally, it has now become more routine to perform high‐throughput sequencing on antibody repertoires to also directly discover antibodies. In this review, we provide an overview of the progress in this field to date and show how high‐throughput screening and sequencing are converging to deliver powerful new workflows for monoclonal antibody discovery and engineering.
In this review, we provide an overview of the progress on how high‐throughput screening and sequencing are converging to deliver powerful new workflows for monoclonal antibody discovery and engineering.
Molecular analysis of circulating tumor cells (CTCs) at single-cell resolution offers great promise for cancer diagnostics and therapeutics from simple liquid biopsy. Recent development of massively ...parallel single-cell RNA-sequencing (scRNA-seq) provides a powerful method to resolve the cellular heterogeneity from gene expression and pathway regulation analysis. However, the scarcity of CTCs and the massive contamination of blood cells limit the utility of currently available technologies. Here, we present Hydro-Seq, a scalable hydrodynamic scRNA-seq barcoding technique, for high-throughput CTC analysis. High cell-capture efficiency and contamination removal capability of Hydro-Seq enables successful scRNA-seq of 666 CTCs from 21 breast cancer patient samples at high throughput. We identify breast cancer drug targets for hormone and targeted therapies and tracked individual cells that express markers of cancer stem cells (CSCs) as well as of epithelial/mesenchymal cell state transitions. Transcriptome analysis of these cells provides insights into monitoring target therapeutics and processes underlying tumor metastasis.
The timely arrival of novel materials plays a key role in bringing advances to society, as the pace at which major technological breakthroughs take place is usually dictated by the discovery rate at ...which novel materials are identified within chemical space. High‐throughput experimentation and computation strategy, now widely considered as a watershed in accelerating the discovery and optimization of novel materials in virtually every field, enables simultaneous screening, synthesis and characterization of large arrays of different material classes toward identification of the lead candidates for given system and targeted application. However, the ability to acquire data, through the continued advancement of automation platforms and workflows especially in the field of battery research and development, often outpaces the ability to optimally leverage obtained data for improved decision‐making. Closing this gap inevitably calls for adapted algorithms, development of reliable predictive models and enhanced integration with machine learning, deep learning, and artificial intelligence. This Review aims to highlight state‐of‐the‐art achievements along with an assessment of current and future challenges as well as resulting perspectives toward accelerated development of advanced battery electrolytes and their interfaces.
Accelerated discovery and development of novel battery materials, especially electrolytes and their interfaces within battery chemistries of interest, unquestionably call for complementary and synergistic high‐throughput experimentation (HTE) and a high‐throughput virtual screening approach. Well‐designed HTE workflows result in a wealth of experimental datasets as a solid foundation for enhanced technical decision making based on adapted machine learning‐based algorithms and high‐performance computing tools.
- 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.
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.
DNA sequencing continues to decrease in cost with the Illumina HiSeq2000 generating up to 600 Gb of paired-end 100 base reads in a ten-day run. Here we present a protocol for community amplicon ...sequencing on the HiSeq2000 and MiSeq Illumina platforms, and apply that protocol to sequence 24 microbial communities from host-associated and free-living environments. A critical question as more sequencing platforms become available is whether biological conclusions derived on one platform are consistent with what would be derived on a different platform. We show that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.
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.
Genomics is a relatively new scientific discipline, having DNA sequencing as its core technology. As technology has improved the cost and scale of genome characterization over sequencing’s 40-year ...history, the scope of inquiry has commensurately broadened. Massively parallel sequencing has proven revolutionary, shifting the paradigm of genomics to address biological questions at a genome-wide scale. Sequencing now empowers clinical diagnostics and other aspects of medical care, including disease risk, therapeutic identification, and prenatal testing. This Review explores the current state of genomics in the massively parallel sequencing era.
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