Small Labs Research Articles

Abstract A10: CRISPR-AnalyzeR (caR): Web-based, interactive and exploratory analysis and documentation of pooled CRISPR/Cas9 screens

Abstract Since the availability of the CRISPR/Cas9 gene editing technique, pooled CRISPR/Cas9 screens have become a powerful and versatile tool for the investigation of functional genomics in a variety of organisms. Pooled CRISPR/Cas9 screens are cost-effective and do not require lab automation, which allows even small labs to perform genome-wide genetic perturbation screens. The analysis of such screens so far requires in-depth knowledge in either bioinformatics or data analysis as there is a limited availability of end-to-end data analysis pipelines (Li et al., 2014; Winter et al., 2015). Moreover, the explorative analysis of screening data using end-to-end analysis tools often comes with certain limitations. Either these pipelines require sufficient computing power on the user machine or have to be run in command-line mode on dedicated machines, which constrains the usability. A special need for wet-lab scientists is the availability of analysis pipelines which allow them to analyze the screening data on their own, knowing best about potential candidates and the biological background. Here we describe the CRISPR-AnalyzeR (caR), which is a web-based and fully-interactive tool to analyze pooled CRISPR/Cas9 screens meant for wet-lab scientists. caR combines both, end-to-end data analysis and explorative screen analysis that can be accessed via a regular web-browser such as Google Chrome or Firefox from any device - allowing the user to analyze the data in a comfortable, convenient and exploratory way. Moreover, caR uses latest visualization technologies to provide interactive plots and tables, allowing even novice users to analyze their own screens in an exploratory way. caR performs hit-calling using six different analysis methods, which gives the user the power and flexibility to select appropriate follow-up candidates. caR provides an easy-to-follow workflow and guides the user through the whole analysis process, which includes quality control, hit calling, in-depth gene information, data annotation enrichment and downloadable interactive reports to provide a comprehensive documentation. Since caR is a web-based tool, it can be used via a provided web-server at http://crispr-analyzer.dkfz.de or can be downloaded for a local network installation. Citation Format: Jan Winter, Marc Schwering, Benedikt Rauscher, Florian Heigwer, Michael Boutros. CRISPR-AnalyzeR (caR): Web-based, interactive and exploratory analysis and documentation of pooled CRISPR/Cas9 screens [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A10.

A novel plant DNA extraction method using filter paper-based 96-well spin plate.

A simple, low cost and safe method using homemade filter paper-based 96-well spin plates and homemade solutions was developed for high throughput plant DNA extraction to use in molecular marker analysis. A low cost and safe method was developed for high throughput extraction of plant DNA for molecular marker analysis. In this method, we describe a simple way to prepare 96-well spin plate using filter paper, a plant material product for DNA binding. Such filter paper-based spin plate can combine with homemade non-toxic buffers for high throughput extraction of plant DNA. We confirmed that filter paper is an efficient solid-phase DNA binding material and comparable to silicon-based glass fiber filters adopted in commercial DNA extraction kits, and that plant DNA extracted by this method can be readily used as template for PCR. The efficacy of this method was also fully demonstrated by molecular marker analysis in segregating populations of tomato. Due to greatly reduced expense compared to commercial kits, this method is of great value for small labs with limited resources.

The Application of Next-Generation Sequencing for Mutation Detection in Autosomal-Dominant Hereditary Hearing Impairment.

Identification of the causative mutation using next-generation sequencing in autosomal-dominant hereditary hearing impairment, as mutation analysis in hereditary hearing impairment by classic genetic methods, is hindered by the high heterogeneity of the disease. Two Swiss families with autosomal-dominant hereditary hearing impairment. Amplified DNA libraries for next-generation sequencing were constructed from extracted genomic DNA, derived from peripheral blood, and enriched by a custom-made sequence capture library. Validated, pooled libraries were sequenced on an Illumina MiSeq instrument, 300 cycles and paired-end sequencing. Technical data analysis was performed with SeqMonk, variant analysis with GeneTalk or VariantStudio. The detection of mutations in genes related to hearing loss by next-generation sequencing was subsequently confirmed using specific polymerase-chain-reaction and Sanger sequencing. Mutation detection in hearing-loss-related genes. The first family harbored the mutation c.5383+5delGTGA in the TECTA-gene. In the second family, a novel mutation c.2614-2625delCATGGCGCCGTG in the WFS1-gene and a second mutation TCOF1-c.1028G>A were identified. Next-generation sequencing successfully identified the causative mutation in families with autosomal-dominant hereditary hearing impairment. The results helped to clarify the pathogenic role of a known mutation and led to the detection of a novel one. NGS represents a feasible approach with great potential future in the diagnostics of hereditary hearing impairment, even in smaller labs.

MetaLIMS, a simple open-source laboratory information management system for small metagenomic labs.

Background: As the cost of sequencing continues to fall, smaller groups increasingly initiate and manage larger sequencing projects and take on the complexity of data storage for high volumes of samples. This has created a need for low-cost laboratory information management systems (LIMS) that contain flexible fields to accommodate the unique nature of individual labs. Many labs do not have a dedicated information technology position, so LIMS must also be easy to setup and maintain with minimal technical proficiency. Findings: MetaLIMS is a free and open-source web-based application available via GitHub. The focus of MetaLIMS is to store sample metadata prior to sequencing and analysis pipelines. Initially designed for environmental metagenomics labs, in addition to storing generic sample collection information and DNA/RNA processing information, the user can also add fields specific to the user's lab. MetaLIMS can also produce a basic sequencing submission form compatible with the proprietary Clarity LIMS system used by some sequencing facilities. To help ease the technical burden associated with web deployment, MetaLIMS options the use of commercial web hosting combined with MetaLIMS bash scripts for ease of setup. Conclusions: MetaLIMS overcomes key challenges common in LIMS by giving labs access to a low-cost and open-source tool that also has the flexibility to meet individual lab needs and an option for easy deployment. By making the web application open source and hosting it on GitHub, we hope to encourage the community to build upon MetaLIMS, making it more robust and tailored to the needs of more researchers.

Assemble CRISPRs from metagenomic sequencing data.

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) allows more specific and efficient gene editing than all previous genetic engineering systems. These exciting discoveries stem from the finding of the CRISPR system being an adaptive immune system that protects the prokaryotes against exogenous genetic elements such as phages. Despite the exciting discoveries, almost all knowledge about CRISPRs is based only on microorganisms that can be isolated, cultured and sequenced in labs. However, about 95% of bacterial species cannot be cultured in labs. The fast accumulation of metagenomic data, which contains DNA sequences of microbial species from natural samples, provides a unique opportunity for CRISPR annotation in uncultivable microbial species. However, the large amount of data, heterogeneous coverage and shared leader sequences of some CRISPRs pose challenges for identifying CRISPRs efficiently in metagenomic data. In this study, we developed a CRISPR finding tool for metagenomic data without relying on generic assembly, which is error-prone and computationally expensive for complex data. Our tool can run on commonly available machines in small labs. It employs properties of CRISPRs to decompose generic assembly into local assembly. We tested it on both mock and real metagenomic data and benchmarked the performance with state-of-the-art tools. The source code and the documentation of metaCRISPR is available at https://github.com/hangelwen/metaCRISPR CONTACT: yannisun@msu.edu.

The anti-Aspergillus drug pipeline: Is the glass half full or empty?

Aspergillosis has emerged as important human mycoses, in view of the ever expanding population at risk. The emergence of resistance to the most commonly used drugs for aspergillosis, the azoles, the mediocre activity, and frequent toxicity of the current antifungal armamentarium, support the need for development of novel antifungals for treatment of this disease. In this minireview, we describe recent efforts by small drug companies and University research labs to develop novel therapies for invasive aspergillus infections. We specifically discuss four small-molecule antifungals (T-2307, E1210/APX001, ASP2397, and F901318) with novel modes-of-action, which are currently entering phase I clinical trials. In addition, we provide a nonexhaustive discussion of some interesting, yet early developments in the quest for improved therapeutic strategies such as (i) novel formulations of amphotericin B including AMB nanoparticle suspensions and AMB-arabinogalactan or AMB-PEG conjugates that show low toxicity and high efficacy in preclinical animal models, (ii) repurposed drugs that synergize with existing antifungals (clozafimine, trichostatin A, MGCD290, geldanamycin, tacrolimus, cyclosporin), (iii) natural products (psoriasin, humidimycin), and (iv) immunotherapy using adoptive transfer of activated immune cells with antifungal activity. We argue that despite the plethora of candidates, the extremely low success rates of drug development leading to clinically useful drugs reinforces the need for continued clinical reliance on mainstream antifungals and their improved derivatives.

ALMA observations of az≈ 3.1 protocluster: star formation from active galactic nuclei and Lyman-alpha blobs in an overdense environment

We exploit ALMA 870um observations to measure the star-formation rates (SFRs) of eight X-ray detected Active Galactic Nuclei (AGNs) in a z~3.1 protocluster, four of which reside in extended Ly-alpha haloes (often termed Ly-alpha blobs: LABs). Three of the AGNs are detected by ALMA and have implied SFRs of ~220-410~M_sun/yr; the non detection of the other five AGNs places SFR upper limits of <210 M_sun/yr. The mean SFR of the protocluster AGNs (~110-210 M_sun/yr) is consistent (within a factor of ~0.7-2.3) with that found for co-eval AGNs in the field, implying that galaxy growth is not significantly accelerated in these systems. However, when also considering ALMA data from the literature, we find evidence for elevated mean SFRs (up-to a factor of ~5.9 over the field) for AGNs at the protocluster core, indicating that galaxy growth is significantly accelerated in the central regions of the protocluster. We also show that all of the four protocluster LABs are associated with an ALMA counterpart within the extent of their Ly-alpha emission. The SFRs of the ALMA sources within the LABs (~150-410 M_sun/yr) are consistent with those expected for co-eval massive star-forming galaxies in the field. Furthermore, the two giant LABs (with physical extents of >100 kpc) do not host more luminous star formation than the smaller LABs, despite being an order of magnitude brighter in Ly-alpha emission. We use these results to discuss star formation as the power source of LABs.

From Soda Bottles To Super Labs: An Analysis Of North America's Dual Methamphetamine Production Networks

This article uses the global production network perspective to analyze the various actors involved in the production and distribution of methamphetamine in North America. The paper analyzes the organization and operation of the two dominant network typologies at work in the United States: small toxic labs and Mexican drug‐trafficking organizations. Particular emphasis is placed on how the networks have adapted to changes in the regulatory context in which they operate. I conclude with potential policy recommendations for curtailing methamphetamine production within both networks.

Is Mississippi's prescription-only precursor control law a prescription to decrease the production and raise the price of methamphetamine?

BackgroundIn 2010, Mississippi became the second state to require a prescription to purchase pseudoephedrine-based medications. Proponents of “prescription-only” laws argue that they are necessary to disrupt methamphetamine markets, but critics note the costs to legal consumers of cold medications may offset some of the laws’ intended benefits. ObjectiveWe evaluated the effect of prescription-only restrictions for methamphetamine precursors on state-level methamphetamine lab seizures and methamphetamine prices. MethodsWe used a synthetic control approach to create a control state comparable to Mississippi and then used permutation testing to determine if the resulting difference was statistically significant. ResultsWe found that Mississippi's prescription-only law removed 2637 small methamphetamine labs in the two years after the law became effective, which represents a 77% reduction in small labs relative to the synthetic counterfactual. We found no evidence that the law impacted methamphetamine prices. ConclusionWe conclude that while prescription-only laws can reduce the number of domestic small methamphetamine labs in operation, methamphetamine availability is unlikely to be materially impacted.

A simple, specific high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin in cell culture medium

A simple, specific, high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin was developed for directly measuring melatonin in cell culture medium with 10% FBS. This assay adopts a commercial monoclonal melatonin antibody and melatonin–HRP conjugate, so it can be applied in multiple labs rapidly with low cost compared with commercial RIA and ELISA kits. In addition, the procedure is much simpler with only four steps: 1) sample/conjugate incubation, 2) plate washing, 3) TMB color reaction and 4) reading of results. The standards of the assay cover a wide working range from 100pg/mL to 10ng/mL. The sensitivity was 68pg/mL in cell culture medium with 10% FBS and 26pg/mL in PBS with as little as 25μL sample volume. The recovery of melatonin from cell culture medium was 101.0%. The principal cross-reacting compound was 5-methoxytryptophol (0.1%). The variation coefficients of the assay, within and between runs, ranged between 6.68% and 15.76% in cell culture medium. The mean linearity of a series diluted cell culture medium sample was 105% (CV=5%), ranging between 98% and 111%, y=5.5263x+0.0646, R2=0.99. The assay enables small research and teaching labs to reliably measure this important neurohormone.

Analytical Interference, Who Should Assess Kits?

Analytical Interference, Who Should Assess Kits?

Handling big data of online social networks on a small machine

Dealing with big data in computational social networks may require powerful machines, big storage, and high bandwidth, which may seem beyond the capacity of small labs. We demonstrate that researchers with limited resources may still be able to conduct big-data research by focusing on a specific type of data. In particular, we present a system called MPT (Microblog Processing Toolkit) for handling big volume of microblog posts with commodity computers, which can handle tens of millions of micro posts a day. MPT supports fast search on multiple keywords and returns statistical results. We describe in this paper the architecture of MPT for data collection and phrase search for returning search results with statistical analysis. We then present different indexing mechanisms and compare them on the microblog posts we collected from popular online social network sites in mainland China.

GASS: genome structural annotation for Eukaryotes based on species similarity.

BackgroundWith the development of high-throughput sequencing techniques, more and more genomes were sequenced and assembled. However, annotating a genome’s structure rapidly and expressly remains challenging. Current eukaryotic genome annotations require various, abundant supporting data, such as: species-specific and cross-species protein sequences, ESTs, cDNA and RNA-Seq data. Collecting those data and merging their analytical results to achieve a consistent complete annotation is a complex, time and cost consuming task.ResultsIn our study, we proposed a fast and easy-to-use computational tool: GASS (Genome Annotation based on Species Similarity). It annotates a eukaryotic genome based on only the annotations from another similar species. With aligning the exons’ sequences of an annotated similar species to the un-annotated genome, GASS detects the optimal transcript annotations with a shortest-path model. In our study, GASS was used to achieve the rhesus annotations based on the human annotations. The produced annotations were evaluated by comparing them to the two existing rhesus annotation databases (RefSeq and Ensembl) directly and being aligned with three RNA-Seq data of rhesus. The experiment results showed that more than 65% RefSeq exons and splicing junctions were exactly found by GASS. GASS’s sensitivity was higher than RefSeq’s, and was close to Ensembl’s. GASS had higher specificities than Ensembl at gene, transcript, exon and splicing junction levels. We also found the mis-assemblies of rheMac3 genome, which led to the 2 bp shifts in annotating position on exons’ boundary and then the incomplete splicing canonical sites in Refseq annotations. These detections were further supported by various data sources.ConclusionsGASS quickly produces structural genome annotations in sufficient abundance and accuracy. With simple and rapid running of GASS, small labs can create quick views of genome annotations for an un-annotated species, without the necessity to create, collect, analyze and synthesize extra various data sources, or wait several months for the annotations from professional organizations. GASS can be applied to many study occasions, such as the analysis of RNA-Seq datasets from the unannotated species whose genome drafts are available but the annotations are not.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1353-3) contains supplementary material, which is available to authorized users.

Astronomy for astronomical numbers: A worldwide massive open online class

&lt;p&gt;Astronomy: State of the Art is a massive, open, online class (MOOC) offered through Udemy by an instructional team at the University of Arizona. With nearly 24,000 enrolled as of early 2015, it is the largest astronomy MOOC available. The astronomical numbers enrolled do not translate into a similar level of engagement. The content consists of 14 hours of video lecture, nearly 1,000 Powerpoint slides, 250 pages of background readings, and 20 podcast interviews with leading researchers. Perhaps in part because of the large amount of course content, the overall completion rate is low, about 3%. However, this number was four times higher for an early cohort of learners who were selected to have a prior interest in astronomy and who took the class in synchronous mode, with new content being added every week. Completion correlates with engagement as measured by posts to the online discussion board. For a subset of learners, social media like Facebook and Twitter provide an additional, important mode of engagement. For the asynchronous learners who have continuously enrolled for the past 15 months, those who complete the course do so quickly, with few persisting longer than two months. The availability of a free completion certificate had no impact of completion rates when it was added midway through the period of data analyzed in this paper. This experiment informs a new offering of an enhanced version of this MOOC via Coursera, along with a co-convened “flipped” introductory astronomy class at the University of Arizona, where the video lectures will be online and class time will be used exclusively for small group labs and hands-on activities. Despite their typically low completion rates, MOOCs have the potential to add significantly to public engagement with science, and they attract a worldwide audience. &lt;/p&gt;

A Simple and Efficient Method for Preparing Cell Slides and Staining without Using Cytocentrifuge and Cytoclips.

Cell staining is a necessary and useful technique for visualizing cell morphology and structure under a microscope. This technique has been used in many areas such as cytology, hematology, oncology, histology, virology, serology, microbiology, cell biology, and immunochemistry. One of the key pieces of equipment for preparing a slide for cell staining is cytology centrifuge (cytocentrifuge) such as cytospin. However, many small labs do not have this expensive equipment and its accessory, cytoclips (also expensive relatively), which makes them difficult to study cell cytology. Here we present an alternative method for preparing a slide and cell staining in the absence of a cytocentrifuge (and cytoclips). This method is based on the principle that a regular cell centrifuge can be used to concentrate cells harvested from cell culture and then deposit the concentrated cell suspension to a slide evenly by using a cell spreader, followed by cell staining. The method presented is simple, rapid, economic, and efficient. This method may also avoid a possible change in cell morphology induced by cytocentrifuge.

Validation of rapid DNA extraction protocol and their effectiveness in marker assisted selection in crop plants

In the present study, a simple and ultra quick protocol for extraction of genomic DNA was successfully validated in four different crops namely rice, wheat, lentil and Indian mustard. The method is simple and involves only two steps, it does not involve use of liquid nitrogen and other expensive and toxic reagents, and is highly suited for analyzing large number of samples within a short span of time. This makes it very attractive for use in MAS even in smaller labs where no sophisticated facilities are available.

Metrology Needs for Predicting Concrete Pumpability

With the increasing use of pumping to place concrete, the development and refinement of the industry practice to ensure successful concrete pumping are becoming important needs for the concrete construction industry. To date, research on concrete pumping has been largely limited to a few theses and research papers. The major obstacle to conduct research on concrete pumping is that it requires heavy equipment and large amounts of materials. Thus, developing realistic and simple measurement techniques and prediction tools is a financial and logistical challenge that is out of reach for small research labs and many private companies in the concrete construction industry. Moreover, because concrete pumping involves the flow of a complex fluid under pressure in a pipe, predicting its flow necessitates detailed knowledge of the rheological properties of concrete, which requires new measurement science. This paper summarizes the technical challenges associated with concrete pumping and the development in concrete pumping that have been published in the technical literature and identifies future research needed for the industry to develop best practices for ensuring successful concrete pumping in the field.

Phenol-stacked carbon nanotubes: A new approach to genomic DNA isolation from plants.

Extraction of intact quality DNA from plant tissues, especially those rich in secondary metabolites, is often challenging. Literally, hundreds of different DNA isolation protocols from various plant species have been published over the last decades. Although many commercial DNA isolation kits are convenient and designed to be safe, their cost and availability cause limitations in small molecular labs in many developing countries. In nearly all protocols and DNA isolation kits, phenol and chloroform are used to precipitate various classes of impurities. However, phenol is partially soluble in water, resulting in the co-existence of proteins in upper (aqueous) phases. This phenomenon results in the contamination of the nucleic acids and low quality DNA. Nanotechnology advances have helped many areas of molecular biology such as the development of new diagnosis and purification kits. In this study, for the first time, we report a different approach to isolate DNA from plants based on carbon nanotubes (CNTs). The results show that the phenol reagent stack on CNTs can effectively remove proteins, polysaccharides and other polyphenol constituents. The A260/A280nm absorbance ratios of isolated DNA samples were 1.9 and 1.8 for chamomile and opium plants, respectively, indicating the high purity of the isolated DNA. DNA yield was more than two times the standard Doyle and Doyle method. Furthermore, the isolated DNA proved amenable to PCR amplification, using Random Amplified Polymorphic DNA (RAPD) analysis.

Next-generation sequencing of 16S ribosomal RNA gene amplicons.

One of the major questions in microbial ecology is “who is there?” This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is “who is there?” This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.