Small Research Laboratories Research Articles

Cryopreservation of Large Number of Human Hematopoietic Cells – A Model for Research Space with Limited Resources

Unlike cell lines, cells from primary human tissues are valuable, requiring storage for long-term use. Large number of primary cells require the most efficient method of shipment, when relevant, and cryopreservation to preserve the viability and function. Shipping of cells could be determined by the specific experimental question. These issues are particularly important for primary cells such as hematopoietic stem cells, which cannot expand in vitro. There is minimum issues when cryopreserving relatively small number of cells from tissues such as umbilical cord blood and bone marrow aspirates. However, cryopreservation of >200 × 109 primary cells comes with challenges to identify the most efficient method for long-term storage. Here we report on processes to achieve efficient cell health and recovery of hematopoietic cells from mobilized peripheral blood cells (MPBs). We also determined overnight shipment of MPBs led to overall outcomes when shipped in the cold, as compared to room temperature. We found that cryopreservation of 50 × 106 cells in 2 mL led to maximum recovery of hematopoietic cell subsets. The implication for these conditions to achieve efficient long-term storage is discussed. These methods will benefit small research laboratories such as those at academic settings and start-up companies with limited resources.

An inexpensive semi-automated sample processing pipeline for cell-free RNA extraction.

Despite advances in automated liquid handling and microfluidics, preparing samples for RNA sequencing at scale generally requires expensive equipment, which is beyond the reach of many academic laboratories. Manual sample preparation remains a slow, expensive and error-prone process. Here, we describe a low-cost, semi-automated pipeline to extract cell-free RNA using one of two commercially available, inexpensive and open-source robotic systems: the Opentrons OT1.0 or OT2.0. Like many RNA isolation protocols, ours can be decomposed into three subparts: RNA extraction, DNA digestion and RNA cleaning and concentration. RT-qPCR data using a synthetic spike-in confirms comparable RNA quality to the gold standard, manual sample processing. The semi-automated pipeline also shows improvement in sample throughput (+12×), time spent (-11×), cost (-3×) and biohazardous waste produced (-4×) compared with its manual counterpart. This protocol enables cell-free RNA extraction from 96 samples simultaneously in 4.5 h; in practice, this dramatically improves the time to results, as we recently demonstrated. Importantly, any laboratory already has most of the parts required (manual pipette and corresponding tips and kits for RNA isolation, cleaning and concentration) to build a semi-automated sample processing pipeline of their own and would only need to purchase or three-dimensionally print a few extra parts (US$5.5 K-12 K in total). This pipeline is also generalizable for many nucleic acid extraction applications, thereby increasing the scale of studies, which can be performed in small research laboratories.

Technical upgrade of an open-source liquid handler to support bacterial colony screening.

The optimization of genetically engineered biological constructs is a key step to deliver high-impact biotechnological applications. The use of high-throughput DNA assembly methods allows the construction of enough genotypic variants to successfully cover the target design space. This, however, entails extra workload for researchers during the screening stage of candidate variants. Despite the existence of commercial colony pickers, their high price excludes small research laboratories and budget-adjusted institutions from accessing such extensive screening capability. In this work we present COPICK, a technical solution to automatize colony picking in an open-source liquid handler Opentrons OT-2. COPICK relies on a mounted camera to capture images of regular Petri dishes and detect microbial colonies for automated screening. COPICK's software can then automatically select the best colonies according to different criteria (size, color and fluorescence) and execute a protocol to pick them for further analysis. Benchmark tests performed for E. coli and P. putida colonies delivers a raw picking performance over pickable colonies of 82% with an accuracy of 73.4% at an estimated rate of 240 colonies/h. These results validate the utility of COPICK, and highlight the importance of ongoing technical improvements in open-source laboratory equipment to support smaller research teams.

A novel accelerated fatigue testing system for pulsatile applications of cardiac devices using widely translatable cam and linkage-based mechanisms.

Fatigue testing of mechanical components is important for designing safe implantable medical prosthetics, and accelerated systems can be used to increase the speed of evaluation. We developed a platform for accelerated testing of linear force applications of cardiac devices, called the Fatigue Acceleration System Tester (FAST). FAST operates using a core translation mechanism, converting motor-driven rotary motion to linear actuation. The advantages of using this mechanism include 40x rate increases with largely 3D-printed components, versatility based on modular design paradigms, and accessible manufacturability with 3D-printable forms, enabling access for small and large research laboratories alike. FAST has been crucial in informing our designs for continuing device development. Over two fatigue cycle courses of 52 and 110 days, the motor cycled at rotational frequencies up to 1500rpm, 43 times faster than those experienced in a typical heart and equating to approximate life cycles of five and ten years, respectively. In designing FAST, our goal was to accessibly bring a strong mechanical basis to study the long-term effects of repeated loading, and we present a design that can be applied across many industries to not only evaluate fatigue performance, but also generate any cycling linear motion.

Comparative Study of the Selective Sorption of Organic Dyes on Inorganic Materials—A Cost-Effective Method for Waste Treatment in Educational and Small Research Laboratories

Educational and research laboratories often produce relatively small amounts of highly diverse organic wastes. Treating waste can contribute significantly to the cost of running laboratories. This study introduced a simple and economical waste management system such that readily available used chromatography-grade inorganic materials, such as silica and alumina (basic and acidic), are utilized to treat remnant dye solutions and solution wastes from educational and small research laboratories. To recycle the adsorbents, they were heated to 600 °C, where the adsorbates were combusted. The results showed that acidic alumina is an effective adsorbent material for azo dyes and anionic dyes/stains, as well as textile dyes, with a 98 to 100% removal efficiency. Furthermore, alumina and silica possess excellent regeneration properties, where the dye removal efficiency of the materials was retained after regeneration at 600 °C. The adsorption properties of the materials were compared with those of aliginite and activated biomass from coffee grounds. Kinetic and thermodynamic studies of the sorption processes on the different materials were carried out. Overall, the inorganic materials used were efficient at removing contaminating remnant organic dyes stemming from educational and small research laboratories.

Evaluation of Hepatoprotective effect of Phyllanthus niruri in experimental model of NAFLD

Background and AIM: Potential utilization of medicine from herbal origin is on the rise due to adverse effects of allopathic medicines. Phyllanthin extracted from Phyllanthus niruri has been shown to be effective in Hepatoprotective effect. Its therapeutic effect in non-alcoholic fatty liver disease (NAFLD) is not much explored. The present study aim was to evaluate the role of phyllanthin in mice fed with methionine choline deficient (MCD) diet. Materials and Methods: Four–five week-old C57lb/6 mice were fed MCD diet for 4 weeks to develop experimental model of NAFLD in small animal research laboratory, PGIMER, Chandigarh. Phyllanthin extract and commercially available phyllanthin was used to assess the impact on NAFLD animal model. After recovery animal weight, liver function test, and lipid profile were measured. Data were showed as mean and standard deviation. Statistical analysis was executing using SPSS v21. Results: Data revealed that administration of 200mg/kg and 400mg/kg crude extract significantly reduced (P<0.0001) bilirubin levels in MCDD mice (0.69±0.14 and 0.85±0.14) respectively. Administration of 400mg/kg phyllanthin significantly reduced SGOT in MCDD mice (23.3±11.87 vs. 50.17±7.68; P=0.002). Administration of 200mg/kg and 400 mg/kg crude extract significantly reduced SGPT in MCDD mice (29.4±14.39 and 21.80±6.12 respectively) (P<0.0001). Similarly, plant phyllanthin improved lipid profile in MCD animals. The plant phyllanthin does not have adverse effect on body weight. Conclusion: Out of the two test trails as plant phyllanthin and commercial phyllanthin, the former suggested hepatoprotective action, as it causes the reversal of damage in the hepatic profile due to NAFLD.

RNAdetector: a free user-friendly stand-alone and cloud-based system for RNA-Seq data analysis

BackgroundRNA-Seq is a well-established technology extensively used for transcriptome profiling, allowing the analysis of coding and non-coding RNA molecules. However, this technology produces a vast amount of data requiring sophisticated computational approaches for their analysis than other traditional technologies such as Real-Time PCR or microarrays, strongly discouraging non-expert users. For this reason, dozens of pipelines have been deployed for the analysis of RNA-Seq data. Although interesting, these present several limitations and their usage require a technical background, which may be uncommon in small research laboratories. Therefore, the application of these technologies in such contexts is still limited and causes a clear bottleneck in knowledge advancement.ResultsMotivated by these considerations, we have developed RNAdetector, a new free cross-platform and user-friendly RNA-Seq data analysis software that can be used locally or in cloud environments through an easy-to-use Graphical User Interface allowing the analysis of coding and non-coding RNAs from RNA-Seq datasets of any sequenced biological species.ConclusionsRNAdetector is a new software that fills an essential gap between the needs of biomedical and research labs to process RNA-Seq data and their common lack of technical background in performing such analysis, which usually relies on outsourcing such steps to third party bioinformatics facilities or using expensive commercial software.

Effects of storage and leukocyte reduction on the concentration and procoagulant activity of extracellular vesicles in canine packed red cells

To characterize the size and procoagulant activity of extracellular vesicles (EV) that accumulate in canine packed red blood cells (pRBCs) over time and the effect of leukocyte reduction on these characteristics. Prospective cohort study. Private small animal specialty referral hospital and university research laboratories. Ten healthy blood donor dogs. Five pRBCs units were obtained according to standard protocols, and 5 were leukocyte-reduced prior to processing. Platelet-free supernatant from the pRBC units was collected on days 0, 10, 20, 32, and 42. Nanoparticle tracking analysis was performed to determine the size and concentration of EVs. Thrombin generation associated with phosphatidylserine-positive EVs was determined using a capture assay. Factor Xa generation associated with phosphatidylserine-positive EVs and tissue factor-positive EVs was measured in a subset of EVs isolated by centrifugation of the supernatant at 20,000 × g. R package nparLD and the Mann-Whitney U-test were used to determine the effect of duration of storage and the effect of leukocyte reduction, respectively. Small (mean<125nm) procoagulant EVs accumulated over time, with significant increases occurring on or after day 20 in both non-leukocyte reduced and leukocyte-reduced units. The procoagulant activity of the EVs was due to phosphatidylserine, not tissue factor. Increases in EV concentration and procoagulant activity occurred earlier in non-leukocyte reduced units. Extracellular vesicle accumulation and procoagulant activity were not decreased at any individual time point by leukocyte reduction. Further studies characterizing and determining the clinical relevance of small procoagulant EVs in pRBCs are warranted.

Scaling pulser output parameters for standard and dry brick configurations

The building block of the linear transformer driver pulser, known as a brick, can be thought of as the smallest, independent unit from which the entire pulser is built. In a ``standard'' brick configuration, two capacitors and a spark-gap switch are connected in series and are oil insulated. Such a brick could be assembled, for example, from two 80-nF, 100-kV capacitors and a 200-kV spark-gap switch. A single brick in this configuration is capable of generating a current pulse with up to 50 kA of peak current and a rise time on the order of 100 ns, after the capacitors have been discharged at a combined output voltage of 200 kV (twice the bipolar charge voltage). By contrast, in a ``dry'' brick configuration, the two capacitors are placed in parallel and a multigap, multichannel ``ball'' switch follows them in series. The dry brick is epoxy insulated, and its slightly different configuration allows it to have a peak output current of up to 100 kA, after being discharged at a total output voltage of 100 kV (equal to the unipolar charge voltage). In this paper, we present how these two different brick topologies, either oil insulated (standard) or epoxy insulated (dry), affect a pulser's output parameters, where the pulser has been constructed by connecting the outputs of $N$ bricks together in parallel. We find that in many cases, the use of a dry brick configuration results in more current being delivered to the load. We also find that if the time to peak current is not a critical parameter for the experiment, then the lower charging voltage across the switch and the absence of insulating oil make the dry brick an excellent alternative to the standard brick, especially for smaller research laboratories with limited maintenance staff.

Experimental Evaluation of a 3D-Printed Fluidic System for a Directional Anemometer.

An evolution of a previously proposed anemometer capable of detecting both the magnitude and the direction of the wind on a plane is proposed. The device is based on a recently formalized principle, consisting of combining the differential pressures measured across distinct diameters of a cylinder to estimate the wind velocity and incidence angle. Differently from previous sensors based on the same principle, the proposed anemometers use 3D printing to fabricate the channel structure that calculates the pressure combination in the fluidic domain. Furthermore, commercial sensors with low power consumption are used to read the two pressures that result from the fluidic processing. The whole fabrication procedure requires inexpensive equipment and can be adopted by small enterprises or research laboratories. Two original channel structures, predicted by previous theoretical work but never experimentally validated, are proposed. The results of detailed experiments performed in a wind tunnel are reported.

WarmStart colorimetric RT-LAMP for the rapid, sensitive and specific detection of Enteroviruses A-D targeting the 5'UTR region.

The aim of the present study was to develop a colorimetric LAMP assay for the detection of enteroviruses belonging to species A-D targeting the 5' untranslated region (5' UTR) of enteroviruses genome. The RNA was converted to cDNA by the reverse transcriptase and then amplified via LAMP by the WarmStart®Bst DNA polymerase, simultaneously in a single reaction tube, so we shortened the reaction time to 50min. The sensitivity of the assay regarding Enterovirus B, C and D was determined to be 0·30 CCID50 assay-1 while the sensitivity for Enterovirus A was 3·00 CCID50 assay-1 . The assay demonstrated high specificity and sensitivity for the detection of 45 reference strains of Enteroviruses A-D and validated on 20 clinical isolates. This assay can be used as a diagnostic tool for the rapid, sensitive and specific detection of enteroviruses, easily implemented in small clinical and research laboratories since LAMP amplicons were visualized by colour changes eliminating the requirement for post-amplification processing steps. We developed a colorimetric assay ideal for field situations for the detection of enteroviruses, by targeting the 5' UTR. This assay demonstrated high specificity and sensitivity, based on its performance on 45 EV A-D reference strains, on 20 EV B clinical isolates and on three non-enteroviral RNA viruses.

Laboratory information management software for engineered mini-protein therapeutic workflow

BackgroundProtein based therapeutics are one of the fastest growing classes of novel medical interventions in areas such as cancer, infectious disease, and inflammation. Protein engineering plays an important role in the optimization of desired therapeutic properties such as reducing immunogenicity, increasing stability for storage, increasing target specificity, etc. One category of protein therapeutics is nature-inspired bioengineered cystine-dense peptides (CDPs) for various biological targets. These engineered proteins are often further modified by synthetic chemistry. For example, candidate mini-proteins can be conjugated into active small molecule drugs. We refer to modified mini-proteins as “Optides” (Optimized peptides). To efficiently serve the multidisciplinary lab scientists with varied therapeutic portfolio research goals in a non-commercial setting, a cost effective extendable laboratory information management system (LIMS) is/was needed.ResultsWe have developed a LIMS named Optide-Hunter for a generalized engineered protein compounds workflow that tracks entities and assays from creation to preclinical experiments. The implementation and custom modules are built using LabKey server, which is an Open Source platform for scientific data integration and analysis. Optide-Hunter contains a compound registry, in-silico assays, high throughput production, large-scale production, in vivo assays and data extraction from a specimen-tracking database. It is used to store, extract, and view data for various therapeutics projects. Optide-Hunter also includes external processing stand-alone software (HPLCPeakClassifierApp) for automated chromatogram classification. The HPLCPeakClassifierApp is used for pre-processing of HPLC data prior to loading to Optide-Hunter. The custom implementation is done using data transformation modules in R, SQL, javascript, and java and is Open Source to assist new users in customizing it for their unique workflows. Instructions for exploring a deployed version of Optide-Hunter can be found at https://www.labkey.com/case%20study/optide-hunterConclusionThe Optide-Hunter LIMS system is designed and built to track the process of engineering, producing and prioritizing protein therapeutic candidates. It can be easily adapted and extended for use in small or large research laboratories where multidisciplinary scientists are collaborating to engineer compounds for potential therapeutic or protein science applications. Open Source exploration of Optide-Hunter can help any bioinformatics scientist adapt, extend, and deploy an equivalent system tailored to each laboratory’s workflow.

WarmStart colorimetric LAMP for the specific and rapid detection of HPV16 and HPV18 DNA

Background and objectivesPersistent infection with High-Risk HPV genotypes is the principal cause for the development of cervical cancer with HPV16 and HPV18 to be the most frequently identified HPV genotypes observed in approximately 70% of cervical cancer cases worldwide. The present study focused on the development of a simple molecular methodology based on WarmStart colorimetric LAMP for the specific identification of HPV16 and HPV18. MethodsThe method was developed by designing LAMP type-specific primer sets that target the E6 gene. The assay was applied using HPV-positive clinical samples along with control cases in order to evaluate the specificity of the newly designed isothermal protocol. In addition, an experimental cutoff value was estimated through reconstitution experiments with HPV-DNA plasmids. LAMP amplicons were visualized by color changes, thus eliminating the requirement for post-amplification processing steps. ResultsThe WarmStart colorimetric LAMP facilitates the isothermal amplification of 10 copies per reaction of both HPV16 and HPV18 DNA, while it exhibits 100% specificity for the detection of the corresponding genotypes in LSIL and HSIL cases. Moreover, the assay demonstrates 100% PPV and 100% NPV. Finally, the sensitivity of conventional PCR with the type-specific LAMP primer sets (B3/F3) for the HPV16, HPV18 DNA detection was 100 copies/reaction and 10 copies/reaction, respectively. ConclusionsThe newly established WarmStart colorimetric LAMP can be considered as a powerful molecular tool that it can be easily implemented in small clinical and research laboratories for a rapid and efficient identification of the most tumorigenic HPV genotypes.

Development of mini-spectrophotometer for determination of plasma glucose

This work demonstrates a novel compact spectrophotometer, “Mini-spectrophotometer”, designed for plasma glucose detection. Unlike conventional spectrophotometer, a light source of the mini spectrophotometer is replaced by a light-emitting diode (LED), and a fabricated polymer-based microwell is used as a cuvette. To validate the downsizing spectrophotometer prototype, the efficiency and reliability for glucose determination are investigated. Using a certain light intensified from LED, the within-run precision of mini-spectrophotometer is found to be 3.9–8.4% while the between-run precision is 6.7–10.8%. The linearity for the quantification of glucose was up to 500 mg dL−1 and the recovery 99.1 ± 3.4% is obtained. The sensitive and selective detection of glucose has been observed; with limit of detection (LOD) of 13.5 mg dL−1 and limit of quantification (LOQ) of 46.2 mg dL−1, respectively. Hemoglobin and triglyceride at high concentration slightly interferes with the proposed instrument. From comparative studies, there are no significant differences between the glucose concentration measured by mini-spectrophotometer and Shimadzu (r2 = 0.9862) or CECIL spectrophotometer (r2 = 0.9853). Using Passing-Bablok regression analysis, the results obtained from mini-spectrophotometer are in close agreement with the two conventional spectrophotometers. Furthermore, using microwell, the sample volume and reagent used in the process can be reduced. The in-house developed mini-spectrophotometer is capable of detecting plasma glucose while maintaining a compact system, demonstrating the potential of high performance, cost-effective, and portable spectrophotometer for clinical chemistry analysis in small routine, research, and teaching medical laboratory technologist.

Genome Engineering of Hybridomas to Generate Stable Cell Lines for Antibody Expression.

From the perspective of academic and small research laboratories, the most common and practical strategy for recombinant expression of full-length monoclonal antibodies is to perform transient plasmid transfection of mammalian cells, resulting in small-scale and limited protein production. The generation of stable antibody producing mammalian cell lines enables larger-scale and consistent expression, however this approach is rarely pursued due to the time-consuming and expensive process of single colony screening and characterization. In order to bridge the gap between the simplicity of transient transfection and consistent production by stable cell lines, we describe a method to stably integrate antibody genes into the endogenous immunogenomic loci of hybridoma cells using CRISPR/Cas9 genome editing. Initially, the antibody variable light (VL) chain is deleted by multiplexed Cas9 cleavage; subsequently, the variable heavy (VH) chain is replaced by a fluorescent reporter gene (mRuby) by Cas9-assisted homology-directed repair (HDR). This cell line is customized by replacing mRuby with a synthetic antibody (consisting of a VL, light constant region and VH) by once again using Cas9-assisted HDR. Due to hybridomas' inherent ability to surface display and secrete antibodies, they provide a suitable host for both the selection and the production process, substantially streamlining the process for stable cell line generation, and thus we refer to this platform as plug-and-(dis)play (PnP) hybridomas.

Development of a validation protocol of enzyme immunoassay kits used for the analysis of steroid hormones in fish plasma

The analysis of steroid hormones 17β-estradiol, testosterone and 11-ketotestosterone in fish plasma provide insight into fish reproductive behavior and development. Typically, steroid hormones are analyzed using gold standard approaches, such as radioimmunoassay and liquid chromatography coupled with tandem mass spectrometry. However, these methods are not always available to small-to-medium research facilities due to their large capital outlay, expensive running costs and ongoing maintenance needs, in addition to requiring a costly radioisotope license and skilled staff to handle and perform the assays. One approach being increasingly used by researchers is commercial enzyme immunoassay (EIA) kits (also known as ELISA kits), analyzed with the use of a UV–vis spectrometer. However, with their increased use, many studies fail to validate their EIA kits or fail to report on the kits sensitivity, accuracy and precision. As such, the aim of this paper is to propose a standardized protocol for reporting EIA kit validation. The proposed protocol was then applied to three fish species in order to demonstrate parallelism, accuracy and precision. Specifically, we tested plasma concentrations of 17β-estradiol for the red and black anemonefish Amphiprion melanopus, and 17β-estradiol, testosterone and 11-keto-testosterone for the barramundi Lates calcarifer and the common coral trout Plectropomus leopardus. Steroid hormones were extracted from fish plasma using a liquid-liquid solvent extraction method with ethyl acetate and n-hexane (50:50 v:v). Following extraction, the plasma dilution curves showed good linearity and parallelism, steroid recoveries were found to be within 90–120% accuracy, and precision variance was <10% in the majority of assays, for the species and steroids measured. Utilization of the proposed standardized validation protocol of EIA kits will assist scientific, fisheries and aquaculture researchers in obtaining reliable steroid hormone measurements, particularly in the field and in smaller research laboratories.

Analysis of Metagenomics Next Generation Sequence Data for Fungal ITS Barcoding: Do You Need Advance Bioinformatics Experience?

During the last few decades, most of microbiology laboratories have become familiar in analyzing Sanger sequence data for ITS barcoding. However, with the availability of next-generation sequencing platforms in many centers, it has become important for medical mycologists to know how to make sense of the massive sequence data generated by these new sequencing technologies. In many reference laboratories, the analysis of such data is not a big deal, since suitable IT infrastructure and well-trained bioinformatics scientists are always available. However, in small research laboratories and clinical microbiology laboratories the availability of such resources are always lacking. In this report, simple and user-friendly bioinformatics work-flow is suggested for fast and reproducible ITS barcoding of fungi.

Study on Fabrication of Ni-5 at.%W Tapes for Coated Conductors from Cylinder Ingots

Ni-5 at.%W (Ni5W) tapes with a strong cube texture were fabricated using the RABiTS technique and by starting from cylindrical shaped ingots. In contrast to a conventional cuboid-shaped ingot, a cylinder shaped ingot has no anisotropy along the axial direction and the resulting tape will therefore, during heavy cold rolling, be characterized by a lower concentration of stress along the edges of the ingot. It can reduce fabrication costs and increase process efficiency. The fraction of cube texture on the surface of the finally recrystallized tapes was investigated using the EBSD technique. It was observed that the fraction of cube texture within 10° from the ideal {001}〈100〉 orientation was ~98% and the fraction of LAGBs was ~90%. The as-obtained tapes have a strong cube texture also very close to the edge of the tape and they would therefore increase the fraction of applicable material while simplifying the heavy rolling process. Accordingly, it suggests that this fabrication method is a good choice to most small scale research laboratories for achieving long length Ni5W tapes for coated conductors with an easy way and a higher fraction of applicable material.

The outsourcing and commercialization of science: Is the lab CEO the future of academic research?

Once upon a time, there was a small academic research laboratory, in an old brick building, on a quaint college campus. Though small, the lab had everything it needed to do quality research and publish in high‐impact journals. There were a handful of keen undergraduate volunteers, a couple of energetic and optimistic grad students, and an old, silverback PI with a modest but sufficiently sized grant. If you peeked through the ivy‐covered windows of this small lab, you would likely spot some large pieces of equipment, like a fridge, fume hood, centrifuge, and computers, and a cluttering of microscopes, beakers, test tubes, and chemical jars. Occasionally, a student would need an instrument or a reagent that was not in the lab. When this happened, the PI grunted and guffawed and then bought the missing item or sent the student down the hall to borrow it from another team. But apart from that, the lab was self‐sufficient and produced a steady stream of papers. This depiction of autonomous research might sound foreign and outdated to many scientists reading this article, but it was once the norm across most universities—just ask that friendly professor emeritus who putters up and down your department hallways. Sure, contemporary research labs are still populated by ambitious students and grizzled PIs; they are still littered with high‐tech tools, flashy computers, and expensive chemicals; and they still try to publish in “big” journals. But in today's fast‐paced academic landscape, producing quality research is no longer a solely in‐house endeavor; rather, it is increasingly dependent on collaborations, alliances, and outsourcing [1]. The days of the lone‐wolf researcher are long gone, and nowhere is this more apparent than in the field of genomics. Anyone who has spent time sequencing or analyzing genomes in the past decade can attest …

(GTG)5 MSP-PCR fingerprinting as a technique for discrimination of wine associated yeasts?

In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure.