Robotic Liquid Handling Research Articles

Streamlining bioactive molecular discovery through integration and automation

The discovery of bioactive small molecules is generally driven via iterative design–make–purify–test cycles. Automation is routinely harnessed at individual stages of these cycles to increase the productivity of drug discovery. Here, we describe recent progress to automate and integrate two or more adjacent stages within discovery workflows. Examples of such technologies include microfluidics, liquid-handling robotics and affinity-selection mass spectrometry. The value of integrated technologies is illustrated in the context of specific case studies in which modulators of targets, such as protein kinases, nuclear hormone receptors and protein–protein interactions, were discovered. We note that to maximize impact on the productivity of discovery, each of the integrated stages would need to have both high and matched throughput. We also consider the longer-term goal of realizing the fully autonomous discovery of bioactive small molecules through the integration and automation of all stages of discovery. The discovery of bioactive small molecules is generally driven via iterative design–make–purify–test cycles. Progress has been made towards the automation and integration of adjacent stages within such discovery workflows, which can increase the efficiency and effectiveness of bioactive small-molecule discovery.

Abstract 1413: Automation of a barcoded ctDNA sequencing assay based on hybrid capture on two robotic pipetting platforms enables processing of large numbers of liquid biopsy samples

Abstract Introduction: Today's ctDNA sequencing assays are either lab-developed tests (LDTs) and offered as centralized service by CLIA labs, or distributed kits (Qiagen, Roche Avenio ctDNA kit) that require manual processing of samples. The AVENIO ctDNA assays (RUO, Roche Molecular Solutions, Pleasanton CA, USA) are based on the hybrid capture technology that utilizes molecular barcoding and detects four mutation classes (SNV, IDels, CNVs, Fusions). The current manual presequencing workflow requires three full working days. With the recent progress in sequencing of longitudinal liquid biopsy samples there is a clear need for automation of the workflow. Here, we present two automated workflows on the Perkin Elmer NGS Sciclone and a modified Hamilton Star. Methods: Batches of 24 samples of 50ng genomic DNA and 24 samples of 50ng DNA from Horizon cell line blends (HD70, HD731, HD733, HD734) were tested in microtiter plates using manual and automated workflows for comparison. AVENIO cfDNA extraction kit and MagnaPure 96 (MP96) were used for manual and automated extractions of 2 mL of plasma. The AVENIO ctDNA assay was used for manual processing of library prep and target enrichment according to the published instructions for use. Two pipetting robots were used for an automated version of the AVENIO ctDNA workflow. The Perkin Elmer Sciclone has a 96 pipetting head and can process 96 samples in 2.5 days. The modified Hamilton Star has a 8 pipetting head and can process 24 samples in 3 days. After library prep and target enrichment, pools of 12 samples were run on the NextSeq. Sequencing QC parameters were analyzed with focus on on-target rate, duplication rate, deduped depth and insert sizes. Results: An automated workflow for the commercially available AVENIO ctDNA assay was successfully established on both the Perkin Elmer Sciclone and modified Hamilton Star. The main sequencing QC parameters were very similar between Sciclone and modified Star. There was no difference in the somatic calls for the HD cell lines from 5% AF (HD730, 731) down to 2% AF (HD733) and 1.3% AF (HD734) for the manual workflow compared to the two automated workflows. Finally, two sets of 24x 2mL plasma samples from 24 patients with metastatic CRC were processed using (a) the manual cfDNA extraction and AVENIO ctDNA workflow, and (b) automated cfDNA extraction (MP96) and automated workflow on the Sciclone. No major difference was observed between the sequencing QC parameters. Conclusions: We have successfully automated the AVENIO ctDNA workflow on both the Perkin Elmer Sciclone and a modified Hamilton Star. Performance characteristics are very similar on both platforms with respect to on-target and duplication rates, deduped depth and insert sizes. The Sciclone is a widely available pipetting robotic liquid handler and can support processing between 8-192 samples per week with one operator. Citation Format: Maria Lange, Sandra Siemann, Corinna Woestmann, Sylvie McNamara, Mirjam Feldkamp, Anja Blueher, Bernd Hinzmann, Andre Rosenthal. Automation of a barcoded ctDNA sequencing assay based on hybrid capture on two robotic pipetting platforms enables processing of large numbers of liquid biopsy samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1413.

Chromatographic parameter determination for complex biological feedstocks.

The application of mechanistic models for chromatography requires accurate model parameters. Especially for complex feedstocks such as a clarified cell harvest, this can still be an obstacle limiting the use of mechanistic models. Another commonly encountered obstacle is a limited amount of sample material and time to determine all needed parameters. Therefore, this study aimed at implementing an approach on a robotic liquid handling system that starts directly with a complex feedstock containing a monoclonal antibody. The approach was tested by comparing independent experimental data sets with predictions generated by the mechanistic model using all parameters determined in this study. An excellent agreement between prediction and experimental data was found verifying the approach. Thus, it can be concluded that RoboColumns with a bed volume of 200 μL can well be used to determine isotherm parameters for predictions of larger scale columns. Overall, this approach offers a new way to determine crucial model input parameters for mechanistic modelling of chromatography for complex biological feedstocks. © 2018 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:1006–1018, 2018

Design and Development of a Technology Platform for DNA-Encoded Library Production and Affinity Selection

DNA-encoded libraries (DELs) have emerged as an efficient and cost-effective drug discovery tool for the exploration and screening of very large chemical space using small-molecule collections of unprecedented size. Herein, we report an integrated automation and informatics system designed to enhance the quality, efficiency, and throughput of the production and affinity selection of these libraries. The platform is governed by software developed according to a database-centric architecture to ensure data consistency, integrity, and availability. Through its versatile protocol management functionalities, this application captures the wide diversity of experimental processes involved with DEL technology, keeps track of working protocols in the database, and uses them to command robotic liquid handlers for the synthesis of libraries. This approach provides full traceability of building-blocks and DNA tags in each split-and-pool cycle. Affinity selection experiments and high-throughput sequencing reads are also captured in the database, and the results are automatically deconvoluted and visualized in customizable representations. Researchers can compare results of different experiments and use machine learning methods to discover patterns in data. As of this writing, the platform has been validated through the generation and affinity selection of various libraries, and it has become the cornerstone of the DEL production effort at Lilly.

Automated Buildup of Biomimetic Films in Cell Culture Microplates for High-Throughput Screening of Cellular Behaviors.

An automatic method is established for layer-by-layer (LbL) assembly of biomimetic coatings in cell culture microplates using a commercial liquid-handling robot. Highly homogeneous thin films are formed at the bottom of each microwell. The LbL film-coated microplates are compatible with common cellular assays, using microplate readers and automated microscopes. Cellular adhesion is screened on crosslinked and peptide-functionalized LbL films and stem cell differentiation in response to increasing doses of bone morphogenetic proteins (2, 4, 7, 9). This method paves the way for future applications of LbL films in cell-based assays for regenerative medicine and high-throughput drug screening.

High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping

Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting. High-content imaging analysis reveals both dose-dependent and threshold effects during organoid differentiation. Immunofluorescence and single-cell RNA sequencing identify previously undetected parietal, interstitial, and partially differentiated compartments within organoids and define conditions that greatly expand the vascular endothelium. Chemical modulation of toxicity and disease phenotypes can be quantified for safety and efficacy prediction. Screening in gene-edited organoids in this system reveals an unexpected role for myosin in polycystic kidney disease. Organoids in HTS formats thus establish an attractive platform for multidimensional phenotypic screening.

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana.

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or lethality. However, the probability of a synthetic small molecule being bioactive is low; therefore, thousands of molecules must be tested in order to find those of interest. Liquid handling robotics systems are designed to handle large numbers of samples, increasing the speed with which a chemical library can be screened in addition to minimizing/standardizing error. To achieve a high-throughput forward chemical genetics screen of a library of 50,000 small molecules on Arabidopsis thaliana (Arabidopsis), protocols using a bench-top multichannel liquid handling robot were developed that require minimal technician involvement. With these protocols, 3,271 small molecules were discovered that caused visible phenotypic alterations. 1,563 compounds induced short roots, 1,148 compounds altered coloration, 383 compounds caused root hair and other, non-categorized, alterations, and 177 compounds inhibited germination.

Hydrogen-deuterium exchange mass spectrometry reveals folding and allostery in protein-protein interactions.

Hydrogen-deuterium exchange mass spectrometry (HDXMS) has emerged as a powerful approach for revealing folding and allostery in protein-protein interactions. The advent of higher resolution mass spectrometers combined with ion mobility separation and ultra performance liquid chromatographic separations have allowed the complete coverage of large protein sequences and multi-protein complexes. Liquid-handling robots have improved the reproducibility and accurate temperature control of the sample preparation. Many researchers are also appreciating the power of combining biophysical approaches such as stopped-flow fluorescence, single molecule FRET, and molecular dynamics simulations with HDXMS. In this review, we focus on studies that have used a combination of approaches to reveal (re)folding of proteins as well as on long-distance allosteric changes upon interaction.

Scope and limitations of carbohydrate hydrolysis for de novo glycan sequencing using a hydrogen peroxide/metallopeptide-based glycosidase mimetic

Acidic hydrolysis is commonly used as a first step to break down oligo- and polysaccharides into monosaccharide units for structural analysis. While easy to set up and amenable to mass spectrometry detection, acid hydrolysis is not without its drawbacks. For example, ring-destruction side reactions and degradation products, along with difficulties in optimizing conditions from analyte to analyte, greatly limits its broad utility. Herein we report studies on a hydrogen peroxide/CuGGH metallopeptide-based glycosidase mimetic design for a more efficient and controllable carbohydrate hydrolysis. A library of methyl glycosides consisting of ten common monosaccharide substrates, along with oligosaccharide substrates, was screened with the artificial glycosidase for hydrolytic activity in a high-throughput format with a robotic liquid handling system. The artificial glycosidase was found to be active towards most screened linkages, including alpha- and beta-anomers, thus serving as a potential alternative method for traditional acidic hydrolysis approaches of oligosaccharides.

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available "design space" represented by "devices" created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction. Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols. The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Automated Robotic Liquid Handling Assembly of Modular DNA Devices.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available "design space" represented by "devices" created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Controlling cell-free metabolism through physiochemical perturbations

Building biosynthetic pathways and engineering metabolic reactions in cells can be time-consuming due to complexities in cellular metabolism. These complexities often convolute the combinatorial testing of biosynthetic pathway designs needed to define an optimal biosynthetic system. To simplify the optimization of biosynthetic systems, we recently reported a new cell-free framework for pathway construction and testing. In this framework, multiple crude-cell extracts are selectively enriched with individual pathway enzymes, which are then mixed to construct full biosynthetic pathways on the time scale of a day. This rapid approach to building pathways aids in the study of metabolic pathway performance by providing a unique freedom of design to modify and control biological systems for both fundamental and applied biotechnology. The goal of this work was to demonstrate the ability to probe biosynthetic pathway performance in our cell-free framework by perturbing physiochemical conditions, using n-butanol synthesis as a model. We carried out three unique case studies. First, we demonstrated the power of our cell-free approach to maximize biosynthesis yields by mapping physiochemical landscapes using a robotic liquid-handler. This allowed us to determine that NAD and CoA are the most important factors that govern cell-free n-butanol metabolism. Second, we compared metabolic profile differences between two different approaches for building pathways from enriched lysates, heterologous expression and cell-free protein synthesis. We discover that phosphate from PEP utilization, along with other physiochemical reagents, during cell-free protein synthesis-coupled, crude-lysate metabolic system operation inhibits optimal cell-free n-butanol metabolism. Third, we show that non-phosphorylated secondary energy substrates can be used to fuel cell-free protein synthesis and n-butanol biosynthesis. Taken together, our work highlights the ease of using cell-free systems to explore physiochemical perturbations and suggests the need for a more controllable, multi-step, separated cell-free framework for future pathway prototyping and enzyme discovery efforts.

A High-Throughput Mass-Spectrometry-Based Assay for Identifying the Biochemical Functions of Putative Glycosidases.

The most commonly employed glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position in potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a qualitative mass spectrometry-based glycosidase assay amenable to high-throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high-throughput robotic liquid handling system for enzyme substrate screening. Identification of glycosidase biochemical function is achieved through the observation of an appropriate decrease in mass between a potential sugar substrate and its corresponding product by electrospray ionization mass spectrometry (ESI-MS). In addition to screening known glycosidases, the assay was demonstrated to characterize the biochemical function and enzyme substrate competency of the recombinantly expressed product of a putative glycosidase gene from the thermophilic bacterium Thermus thermophilus.

P211 A roadmap for successful robotic automation of next generation sequencing based HLA typing

Aim DNA library preparation for next generation sequencing (NGS) consists of several hundred material transfer and wash steps, which are time consuming, labor intensive, and increase the risk of repetitive strain injury. Robotic liquid handlers can automate and standardize library preparation, but few HLA laboratories have experience with high-complexity robotic platforms. We sought to evaluate the use of robotics for library preparation in conjunction with a NGS based HLA typing assay. Methods A Beckman Coulter Biomek NXp Span-8 robotic liquid handler was acquired along with the vendor supplied robotics method for the Illumina TruSight HLA v2 sequencing panel. The robotics method was first optimized with a series of runs using water, 50% glycerol, 85% ethanol, and AMPure beads. Next, DNA and reagents were run to identify sample viscosity and reagent issues. After each run, issues were communicated to the vendor for method improvement. The finalized method was run on 78 DNA samples over seven runs to assess the quality of normalization, pooling, sequencing, and reproducibility of results. Results Robotic issues were encountered with pipette calibration, labware definitions, proper labware selection, and efficiency. To add complexity, these issues had subtle compounding effects on the overall success of the method, which required that each be addressed individually and in detail. Eleven versions of the method were subsequently produced and tested to ensure robustness and consistency. The finalized method produced consistent high quality sequencing metrics on the NGS typing platform which agreed with the known HLA typing results. Conclusions Detailed documentation of issues by lab staff and clear communication with the vendor allowed for a rapid and iterative improvement of the robotics method. The lessons learned during the implementation process formed a roadmap that allowed for successful implementation of a robotics platform for NGS based HLA typing.

Use of MALDI-MS Combined with Differential Hydrogen-Deuterium Exchange for Semiautomated Protein Global Conformational Screening.

Matrix-assisted laser desorption/ionization (MALDI) coupled with a time-of-flight (TOF) mass-spectrometry (MS) detector is acknowledged to be very useful for analysis of biological molecules. At the same time, hydrogen-deuterium exchange (HDX) is a well-known technique for studying protein higher-order structure. However, coupling MALDI with HDX has been challenging because of undesired back-exchange reactions during analysis. In this report, we survey an approach that utilizes MALDI coupled with an automated sample preparation to compare global conformational changes of proteins under different solution conditions using differential HDX. A nonaqueous matrix was proposed for MALDI sample preparation to minimize undesirable back-exchange. An automated experimental setup based on the use of a liquid-handling robot and automated data acquisition allowed for tracking protein conformational changes as a difference in the number of protons exchanged to deuterons at specified solution conditions. Experimental time points to study the deuteration-labeling kinetics were obtained in a fully automated manner. The use of a nonaqueous matrix solution allowed experimental error to be minimized to within 1% RSD. We applied this newly developed MALDI-HDX workflow to study the effect of several common excipients on insulin folding stability. The observed results were corroborated by literature data and were obtained in a high-throughput and automated manner. The proposed MALDI-HDX approach can also be applied in a high-throughput manner for batch-to-batch higher-order structure comparison, as well as for the optimization of protein chemical modification reactions.

Liquid-handling Lego robots and experiments for STEM education and research.

Liquid-handling robots have many applications for biotechnology and the life sciences, with increasing impact on everyday life. While playful robotics such as Lego Mindstorms significantly support education initiatives in mechatronics and programming, equivalent connections to the life sciences do not currently exist. To close this gap, we developed Lego-based pipetting robots that reliably handle liquid volumes from 1 ml down to the sub-μl range and that operate on standard laboratory plasticware, such as cuvettes and multiwell plates. These robots can support a range of science and chemistry experiments for education and even research. Using standard, low-cost household consumables, programming pipetting routines, and modifying robot designs, we enabled a rich activity space. We successfully tested these activities in afterschool settings with elementary, middle, and high school students. The simplest robot can be directly built from the widely used Lego Education EV3 core set alone, and this publication includes building and experiment instructions to set the stage for dissemination and further development in education and research.

Bead-Extractor Assisted Ready-to-Use Reagent System (BEARS) for Immunoprecipitation Coupled to MALDI-MS

Quantitative protein assays play an important role in the study of biological functions. Immunoassays and mass spectrometry are two main technologies for quantifying proteins in biological samples. The combination of immunoprecipitation (IP) with MALDI technology delivers high assay sensitivity and specificity, but the sample preparation procedure involves multiple washing and transfer steps. These steps can be performed either manually (requiring significant time and labor) or automatically (requiring the purchase of a complex liquid-handling workstation). This bottleneck has limited the widespread adoption of this technology. We present here the Bead-Extractor Assisted ready-to-use Reagent System (BEARS) technology for simplified, low cost protein and peptide immunoprecipitation combined with MALDI-MS detection. All of the reagents are stable during long-term storage and can be prepared in advance. In the BEARS technology, a magnetic-bead extractor is used to handle beads from 96 wells simultaneously. A BEARS-based method was developed for plasma renin activity (PRA) and was evaluated on fifty-three clinical samples. These experiments showed that the BEARS assay had an LOD and linear range comparable to the manual method and an automated iMALDI PRA assay, but was 4-times faster than the manual approach. The BEARS iMALDI results also correlated well with a conventional ELISA PRA assay, with a coefficient of determination of 0.98. The BEARS technology provides convenience and affordability, and extends the use of IP-based mass spectrometry technology to most research and clinical laboratories, including those in developing countries.

A framework for accelerated phototrophic bioprocess development: integration of parallelized microscale cultivation, laboratory automation and Kriging-assisted experimental design

BackgroundEven though microalgae-derived biodiesel has regained interest within the last decade, industrial production is still challenging for economic reasons. Besides reactor design, as well as value chain and strain engineering, laborious and slow early-stage parameter optimization represents a major drawback.ResultsThe present study introduces a framework for the accelerated development of phototrophic bioprocesses. A state-of-the-art micro-photobioreactor supported by a liquid-handling robot for automated medium preparation and product quantification was used. To take full advantage of the technology’s experimental capacity, Kriging-assisted experimental design was integrated to enable highly efficient execution of screening applications. The resulting platform was used for medium optimization of a lipid production process using Chlorella vulgaris toward maximum volumetric productivity. Within only four experimental rounds, lipid production was increased approximately threefold to 212 ± 11 mg L−1 d−1. Besides nitrogen availability as a key parameter, magnesium, calcium and various trace elements were shown to be of crucial importance. Here, synergistic multi-parameter interactions as revealed by the experimental design introduced significant further optimization potential.ConclusionsThe integration of parallelized microscale cultivation, laboratory automation and Kriging-assisted experimental design proved to be a fruitful tool for the accelerated development of phototrophic bioprocesses. By means of the proposed technology, the targeted optimization task was conducted in a very timely and material-efficient manner.

A Cell-Based High-Throughput Method for Identifying Modulators of Alternative Splicing.

Alternative splicing, a key regulatory process of gene expression, is controlled by trans-acting factors that recognize cis-elements in premature RNA transcripts to affect spliceosome assembly and splice site choices. Extracellular stimuli and signaling cascades can converge on RNA binding splicing regulators to affect alternative splicing. Defects in splicing regulation have been associated with various human diseases, and modification of disease-causing splicing events presents great therapeutic promise. Determining splicing regulators and/or upstream modulators has been difficult with low throughput, low sensitivity, and low specificity. IRAS (identifying regulators of alternative splicing) is a novel cell-based high-throughput screening strategy designed specifically to address these challenges and has achieved high throughput, high sensitivity, and high specificity. Here, we describe the IRAS method in detail with a pair of dual-fluorescence minigene reporters that produces GFP and RFP fluorescent signals to assay the two spliced isoforms exclusively. These two complementary mini-gene reporters alter GFP/RFP output ratios in the opposite direction in response to only a true splicing change. False positives from a signal screen do not stimulate opposite changes in GFP/RFP ratios. The reporter pair in conjunction with robotic liquid handlers and arrayed libraries allows IRAS to screen for both positive and negative splicing regulators with high sensitivity and specificity in a high-throughput manner.

Long Fragment Read (LFR) Technology: Cost-Effective, High-Quality Genome-Wide Molecular Haplotyping.

In this chapter, we describe Long Fragment Read (LFR) technology, a DNA preprocessing method for genome-wide haplotyping by whole genome sequencing (WGS). The addition of LFR prior to WGS on any high-throughput DNA sequencer (e.g., Complete Genomics Revolocity™, BGISEQ-500, Illumina HiSeq, etc.) enables the assignment of single-nucleotide polymorphisms (SNPs) and other genomic variants onto contigs representing contiguous DNA from a single parent (haplotypes) with N50 lengths of up to ~1 Mb. Importantly, this is achieved independent of any parental sequencing data or knowledge of parental haplotypes. Further, the nature of this method allows for the correction of most amplification, sequencing, and mapping errors, resulting in false-positive error rates as low as 10-9. This method can be employed either manually using hand-held micropipettors or in the preferred, automated manner described below, utilizing liquid-handling robots capable of pipetting in the nanoliter range. Automating the method limits the amount of hands-on time and allows significant reduction in reaction volumes. Further, the cost of LFR, as described in this chapter, is moderate, while it adds invaluable whole genome haplotype data to almost any WGS process.