Abstract
Laboratory Automation and High-Throughput Chemistry
Automated Gel Size Selection to Improve the Quality of Next-Generation Sequencing Libraries Prepared from Environmental Water Samples
Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High-quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that coprecipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow.
The protocol described in this article enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. Uyaguari-Diaz et al. describe a high-throughput approach to prepare high-quality DNA libraries from freshwater samples that can be applied also to other environmental samples. This study uses an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA is extracted using a commercially available DNA extraction kit, and DNA libraries are prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp are gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrates significant improvements to read length and quality of the sequencing reads (Uyaguari-Diaz, M., et al., J. Vis. Exp.
Current Status and Future Prospects of Point-of-Care Testing around the Globe
In the past half-century, routine central laboratory testing has become increasingly automated and efficient. The majority of clinical chemistry, immunochemistry, and hematology testing is performed using high-throughput instrumentation with sophisticated automation. Microbiology, immunohematology, and molecular diagnostic testing are also becoming increasingly automated. Recent challenges in health care demand new diagnostic solutions worldwide. Point-of-care testing (POCT) offers considerable advantages over central laboratory testing, such as fast and simple specimen handling and simpler sample requirement (no additives and mostly blood from finger stick, as well as urine). No transportation is required, and POCT delivers a short turnaround time of approximately 5 to 15 min. In recent years, POCT has gained ground worldwide. In advanced health care systems, POCT may be beneficial if health or cost-benefits can be established. In resource-poor countries, POCT may be the only means of delivering advanced testing for epidemiologically important diseases, such as tuberculosis or human immunodeficiency virus infection (Abel, G., Exp. Rev. Mol. Diagn.
A High-Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution
Naimuddin and Kubo describe a high-performance platform based on complementary DNA (cDNA) display technology by developing a new modified puromycin linker oligonucleotide. The linker consists of four major characteristics: a ligation site for hybridization and ligation of messenger RNA (RNA) by T4 RNA ligase, a puromycin arm for covalent linkage of the protein, a poly-adenosine site for a longer puromycin arm and purification of protein fusions (optional) using oligo-dT matrices, and a reverse transcription site for the formation of stable cDNA protein fusions whose cDNA is covalently linked to its encoded protein. The linker is synthesized by a novel branching strategy and provides >8-fold higher yield than previous linkers.
This linker enables rapid and highly efficient ligation of mRNA (>90%) and synthesis of protein fusions (~50%–95%) in various cell-free expression systems. Overall, this new cDNA display method provides 10- to 200-fold higher end-usage fusions than previous methods and benefits higher diversity libraries crucial for directed protein/peptide evolution. With the increased efficiency, this system is able to reduce the time for one selection cycle to <8 h and is potentially amenable to high-throughput systems. The authors demonstrate the efficiency of this system for higher throughput selections of various biomolecular interactions and achieve 30- to 40-fold enrichment per selection cycle.
Furthermore, a 4-fold higher enrichment of Flag-tag is obtained from a doped mixture compared with the previous cDNA display method. A three-finger protein library is evolved to isolate superior nanomolar range binding candidates for vascular endothelial growth factor. This method is expected to provide beneficial impact to accelerated drug discovery and proteome analysis (Naimuddin, M., and Kubo, T., ACS Comb. Sci.
Microfluidic Chip Technology and Microreactor Technology
Development of an Automated and Sensitive Microfluidic Device for Capturing and Characterizing Circulating Tumor Cells (CTCs) from Clinical Blood Samples
Current analysis of circulating tumor cells (CTCs) is hindered by suboptimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical biomarkers. In this report, Gogoi et al. validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate, and colorectal cancers using a microfluidic chip that is processed by using an automated staining and scanning system from sample preparation to image processing.
The Celsee system allows for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitates rapid capture of CTCs from blood samples and also allows for downstream characterization of the captured cells by immunohistochemistry, DNA, and mRNA fluorescence in situ hybridization (FISH). In a subset of patients with prostate cancer, the authors compare the technology with a Food and Drug Administration–approved CTC device, Cell-Search, and find a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization (Gogoi, P., et al., PLoS One.
Cell Refractive Index for Cell Biology and Disease Diagnosis: Past, Present and Future
Cell refractive index is a key biophysical parameter that has been extensively studied. It is correlated with other cell biophysical properties, including mechanical, electrical, and optical properties, and not only represents the intracellular mass and concentration of a cell but also provides important insight for various biological models. Measurement techniques developed earlier only measure the effective refractive index of a cell or a cell suspension, providing only limited information on cell refractive index and hence hindering its in-depth analysis and correlation.
Recently, the emergence of microfluidic, photonic, and imaging technologies has enabled the manipulation of a single cell and the 3D refractive index of a single cell down to submicron resolution, providing powerful tools to study cells based on refractive index. In this review, Liu et al. provide an overview of cell refractive index models and measurement techniques, including microfluidic chip–based techniques for the past 50 years; present the applications and significance of cell refractive index in cell biology, hematology, and pathology; and discuss future research trends in the field, including 3D imaging methods, integration with microfluidics, and potential applications in new and breakthrough research areas (Liu, P. Y., et al., Lab Chip
High-Throughput Automation and Systems Biology
The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond
Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction-based techniques, such as the Agilent (Santa Clara, CA, USA) RapidFire system, have emerged and are capable of analyzing samples in <10 s. While dramatically faster than liquid chromatography–coupled MS, an analysis time of 8 to 10 s is still considered relatively slow for full-diversity high-throughput screening (HTS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) offers an alternative for high-throughput MS detection. However, sample preparation and deposition onto the MALDI target, as well as interference from matrix ions, have been considered limitations for the use of MALDI for screening assays.
Haslam et al. describe the development and validation of assays for both small-molecule and peptide analytes using MALDI-TOF coupled with nanoliter liquid handling. Using the JMJD2c histone demethylase and acetylcholinesterase as model systems, the authors generate robust data in a 1536 format and also increase sample deposition to 6144 samples per target. Using these methods, the authors demonstrate that this technology can deliver fast sample analysis time with low sample volume, as well as data comparable to that of current RapidFire assays (Haslam, C., et al., J. Biomol. Screen.
Platform for High-Throughput Antibody Selection Using Synthetically-Designed Antibody Libraries
Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>1020), the practical diversity that can be achieved by transformation of Escherichia coli is limited to about 1010. To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, Batonick et al. generate a scFv phage library using entirely predefined complementarity determining regions (CDRs). The authors use this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target (Batonick, M., et al., N. Biotechnol.
A Cell-Free Expression and Purification Process for Rapid Production of Protein Biologics
Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation, and such systems have been extensively used for high-throughput protein production and screening. Current fluidic systems, however, are not adequate for manufacturing protein biopharmaceuticals. In this report, Sullivan et al. describe the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins.
Using this approach, the authors demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 h. This process is flexible, scalable, and amenable to automation for rapid production at the point of need of proteins with significant pharmaceutical, medical, or biotechnological value (Sullivan, C. J., et al., Biotechnol. J.
Next-Generation Sequencing and Protein Mass Spectrometry for the Comprehensive Analysis of Human Cellular and Serum Antibody Repertoires
Recent developments of high-throughput technologies are enabling the molecular-level analysis and bioinformatic mining of antibody-mediated (humoral) immunity in humans at an unprecedented level. These approaches explore the sequence space of B-cell receptor repertoires using next-generation deep sequencing (BCR-seq), the amino acid identities of antibody in blood using protein mass spectrometry (Ig-seq), or both. Generalizable principles about the molecular composition of the protective humoral immune response are being defined, and as such, the field could supersede traditional methods for the development of diagnostics, vaccines, and antibody therapeutics. Three key challenges remain and have driven recent advances: (1) incorporation of innovative techniques for paired BCR-seq to ascertain the complete antibody variable-domain VH:VL clonotype, (2) integration of proteomic Ig-seq with BCR-seq to reveal how the serum antibody repertoire compares with the antibody repertoire encoded by circulating B cells, and (3) a demand to link antibody sequence data to functional meaning (binding and protection) (Lavinder, J. J., et al., Curr. Opin. Chem. Biol.
Multiplexed Engineering in Biology
Biotechnology is the manufacturing technology of the future. However, engineering biology is complex, and many possible genetic designs must be evaluated to find cells that produce high levels of a desired drug or chemical. Recent advances have enabled the design and construction of billions of genetic variants per day, but evaluation capacity remains limited to thousands of variants per day.
Rogers and Church evaluate biological engineering through the lens of the design-build-test cycle framework and highlight the role that multiplexing has had in transforming the design and build steps. The authors describe a multiplexed solution to the test step that is enabled by new research. Achieving a multiplexed test step will permit a fully multiplexed engineering cycle and boost the throughput of biobased product development by up to a million-fold (Rogers, J. K., and Church, G. M., Trends Biotechnol., in press).
Genetically Encoded Sensors Enable Real-Time Observation of Metabolite Production
Engineering cells to produce valuable metabolic products is hindered by the slow and laborious methods available for evaluating product concentration. Consequently, many designs go unevaluated, and the dynamics of product formation over time go unobserved.
Rogers and Church present a framework for observing product formation in real time without the need for sample preparation or laborious analytical methods. The authors use genetically encoded biosensors derived from small-molecule responsive transcription factors to provide a fluorescent readout that is proportional to the intracellular concentration of a target metabolite. Combining an appropriate biosensor with cells designed to produce a metabolic product allows them to track product formation by observing fluorescence. With individual cells exhibiting fluorescent intensities proportional to the amount of metabolite they produce, high-throughput methods can be used to rank the quality of genetic variants or production conditions. Rogers and Church observed production of several renewable plastic precursors with fluorescent readouts and demonstrate that higher fluorescence is indeed an indicator of higher product titer. Using fluorescence as a guide, the authors identify process parameters that produce 3-hydroxypropionate at 4.2 g/L, 23-fold higher than previously reported. They also report, to their knowledge, the first engineered route from glucose to acrylate, a plastic precursor with global sales of $14 billion. Finally, the authors monitor the production of glucarate, a replacement for environmentally damaging detergents, and muconate, a renewable precursor to polyethylene terephthalate and nylon with combined markets of $51 billion, in real time, demonstrating that the method is applicable to a wide range of molecules (Rogers, J. K., and Church, G. M. Proc. Natl. Acad. Sci. U. S. A.
Advances in Sequencing and Synthetic Biology
Multiplexed Barcoded CRISPR-Cas9 Screening Enabled by CombiGEM
The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. In this study, Wong et al. create a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translate these hits into effective drug combinations.
This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. The authors apply CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they are targeted. The authors validate the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas–based knockouts and RNA interference–based knockdowns. The authors also test small-molecule drug pairs directed against the pairwise hits and show that they exert synergistic antiproliferative effects against ovarian cancer cells.
Wong et al. envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes (Wong, A. S., et al., Proc. Natl. Acad. Sci. U. S. A.
Constructing an Integrated Genetic and Epigenetic Cellular Network for Whole Cellular Mechanism Using High-Throughput Next-Generation Sequencing Data
Epigenetics has been investigated in cancer initiation and development, especially, since the appearance of epigenomics. Epigenetics may be defined as the mechanisms that lead to heritable changes in gene function and without affecting the sequence of genome. These mechanisms explain how individuals with the same genotype produce phenotypic differences in response to environmental stimuli.
Recently, with the accumulation of high-throughput next-generation sequencing (NGS) data, a key goal of systems biology is to construct networks for different cellular levels to explore whole cellular mechanisms. At present, there is no satisfactory method to construct an integrated genetic and epigenetic cellular network (IGECN), which combines NGS omics data with gene regulatory networks (GRNs), microRNA (miRNA) regulatory networks, protein-protein interaction networks (PPINs), and epigenetic regulatory networks of methylation using high-throughput NGS data.
Chen and Li investigate different kinds of NGS omics data to develop a systems biology method to construct an integrated cellular network based on three coupling models that describe genetic regulatory networks, protein-protein interaction networks, miRNA regulatory networks, and methylation regulation. The proposed method is applied to construct IGECNs of gastric cancer and the human immune response to HIV infection, to elucidate human defense response mechanisms. The authors successfully construct an IGECN and validate it by using evidence from literature searches. The integration of NGS omics data related to transcription regulation, protein-protein interactions, and miRNA and methylation regulation has more predictive power than independent data sets. The authors find that dysregulation of MIR7 contributes to the initiation and progression of inflammation-induced gastric cancer; dysregulation of MIR9 contributes to HIV-1 infection to hijack CD4+ T cells through dysfunction of the immune and hormone pathways; dysregulation of MIR139-5p, MIRLET7i, and MIR10a contributes to the HIV-1 integration/replication stage; dysregulation of MIR101, MIR141, and MIR152 contributes to the HIV-1 virus assembly and budding mechanisms; and dysregulation of MIR302a contributes to microvesicle-mediated transfer of miRNAs and dysfunction of nuclear factor–κB signaling pathway in hepatocarcinogenesis.
The coupling dynamic systems of the whole IGECN can allow us to investigate genetic and epigenetic cellular mechanisms via omics data and big database mining and are useful for further experiments in the field of systems and synthetic biology (Chen, B. S., and Li, C. W., BMC Syst. Biol.
Ultra-High-Throughput Sequencing of the Immune Receptor Repertoire from Millions of Lymphocytes
High-throughput sequencing of the variable domains of immune receptors (antibodies and T-cell receptors [TCRs]) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH + VL or TCRβ/δ + TCRα/γ) at the single-cell level for typical samples containing >104 lymphocytes is problematic, because immune receptors comprise two polypeptide chains that are encoded by separate mRNAs.
In this article, McDaniel et al. present a technology that allows rapid and low-cost determination of a paired immune receptor repertoire from millions of cells with high precision (>97%). Flow focusing is used to encapsulate single cells in emulsions containing magnetic beads for mRNA capture. The mRNA transcripts are then reverse-transcribed, physically linked to their partners by overlap extension PCR, and interrogated by high-throughput paired-end Illumina (San Diego, CA, USA) sequencing. This protocol describes the construction and operation of the flow-focusing device in detail, as well as the bioinformatics pipeline used to interpret the data. The entire procedure can be performed by a single researcher in under 12 h of effort per sample (McDaniel, J. R., et al. Nat. Protoc.
Footnotes
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.