Experience of quantity and quality of DNA and RNA extraction from limited pediatric blood samples: A comparative analysis of automated and manual kit-based method.

INTRODUCTION: Optimal DNA and RNA quantity and purity is essential for downstream molecular biology experimentation and to avoid re-processing of sample. Despite availability of different kits and automated systems for nucleic acid isolation there is limited data on their performance evaluation, more so with pediatric blood samples, that are usually compromised in quantity. Hence, we evaluated the performance of automated QIAcube platform using pediatric blood samples in parallel with manual Qiagen extraction kits. MATERIALS AND METHODS: : A total of 500 samples were analyzed based on groups of PBMC and direct blood input. The isolated DNA and RNA were surveyed for quantity and quality tests by spectrophotometric and downstream analysis. RESULTS: : There was no significant difference in the DNA quantity (ng/ul) between manual and automated method based on similar sample input but quality (260/280) was significantly better with the QIAcube platform when direct blood and or PBMCs were used for extraction respectively (1.82 ± 004 Vs. 1.84.002; P-0.000008 and 1.859 ± 005 Vs. 1.843 ± 0.003; P-0.02). Moreover, the standard error mean was low for both quantity and quality in the QIAcube method suggesting uniformity. Comparison of quality assessment by spectrophotometer and qubit fluorimeter showed that QIAcube sheared DNA less (P- 0.038) as compared to manual method (P-0.013). Also, time taken to process the samples in QIAcube was 23% less than the kit-based method. CONCLUSION: Overall analysis of QIAcube platform suggests that it yields more better, uniform, and less-sheared quality of nucleic acid in a relatively less time as compared to manual extraction kits.

Publisher's Note: Continued Publication of Current Issues in Molecular Biology by MDPI.

Current Issues in Molecular Biology (ISSN 1467-3045) was launched in 1999 and has published international and multidisciplinary articles on all aspects of molecular biology spanning from basic mechanisms to applications in fields primarily, but not exclusively, relevant to microbiology and virology [...].

The 2021 Nucleic Acids Research database issue and the online molecular biology database collection.

The 2021 Nucleic Acids Research database Issue contains 189 papers spanning a wide range of biological fields and investigation. It includes 89 papers reporting on new databases and 90 covering recent changes to resources previously published in the Issue. A further ten are updates on databases most recently published elsewhere. Seven new databases focus on COVID-19 and SARS-CoV-2 and many others offer resources for studying the virus. Major returning nucleic acid databases include NONCODE, Rfam and RNAcentral. Protein family and domain databases include COG, Pfam, SMART and Panther. Protein structures are covered by RCSB PDB and dispersed proteins by PED and MobiDB. In metabolism and signalling, STRING, KEGG and WikiPathways are featured, along with returning KLIFS and new DKK and KinaseMD, all focused on kinases. IMG/M and IMG/VR update in the microbial and viral genome resources section, while human and model organism genomics resources include Flybase, Ensembl and UCSC Genome Browser. Cancer studies are covered by updates from canSAR and PINA, as well as newcomers CNCdatabase and Oncovar for cancer drivers. Plant comparative genomics is catered for by updates from Gramene and GreenPhylDB. The entire Database Issue is freely available online on the Nucleic Acids Research website (https://academic.oup.com/nar). The NAR online Molecular Biology Database Collection has been substantially updated, revisiting nearly 1000 entries, adding 90 new resources and eliminating 86 obsolete databases, bringing the current total to 1641 databases. It is available at https://www.oxfordjournals.org/nar/database/c/.

Development of Rapid Pharmacogenomic Testing Assay in a Mobile Molecular Biology Laboratory (2MoBiL).

Pharmacogenomics is rapidly assuming an integral part in modern health care. Still, its broad applicability relies on the feasibility of performing pharmacogenomic testing in all clinical settings, including in remote areas or resource-limited settings with budget restrictions. In this study, we describe the development and feasibility of rapid and reliable pharmacogenomics assays using a portable molecular biology laboratory, namely the 2MoBiL (Mobile Molecular Biology Laboratory). More precisely, we demonstrate that the genotyping of rs4149056, located within SLCO1B1, can be efficiently and reliably performed using the 2MoBiL portable laboratory and conventional benchtop laboratory equipment and a gold standard genotyping method (KASP assay) as directly comparable methodologies. Taking into account the compact size of 2MoBiL, which directly and positively impacts on its portability, and the high accuracy achieved, we conclude that the 2MoBiL-based genotyping method is warranted for further studies in clinical practices at remote areas and resource-limited as well as time-constrained planetary health settings. To contextualize the broader and potential future applications of 2MoBiL, we emphasize that genotyping of a limited set of clinically relevant single-nucleotide polymorphisms is often a common endpoint of genomics and pharmacogenomics discovery and translational research pipeline. Hence, rapid genotyping by 2MoBiL can be an essential catalyst for global implementation of pharmacogenomics and personalized medicine in the clinic. The Clinical Trial Registration number is NCT03093818.

Benchmarking tomographic acquisition schemes for high-resolution structural biology.

Cryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution in principle can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition. Our data is publicly available and might be used to further develop processing routines.

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum.

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

Inconsistencies in some common terms and notations in enzymology: Textbook examples and suggestions.

A number of terms have been defined and inconsistently used across various texts in enzymology which may attract the attention of undergraduate students and instructors. At some places, inconsistent use results in confusions as well as leads to the misuse of these terms. Although it is difficult to resolve such inconsistencies, attempts have constantly been made by international regulatory forums such as International Union of Biochemistry and Molecular Biology (IUBMB) and other similar organizations to abolish the inconsistencies. This article highlights some of the most commonly occurring confusing and inconsistent terms in enzymology. We have gathered shreds of evidences and provide here possible suggestions and caution for readers and instructors, so that mistakes can be minimized and possible guidelines can be prepared for their usage. A few inconsistencies observed and discussed here include different notations of activation energy, differences in terms uncompetitive and noncompetitive in enzyme inhibition, classification of cofactors and definitions of their sub-types, and confusing usage of the enzyme-inhibitor dissociation constant. Besides this, we have also highlighted the inconsistencies in the enzyme nomenclature. © 2018 International Union of Biochemistry and Molecular Biology, 47(2): 140-144, 2019.

Results of the UK NEQAS for Molecular Genetics reference sample analysis.

AIMS: In addition to providing external quality assessment (EQA) schemes, United Kingdom National External Quality Assessment service (UK NEQAS) for Molecular Genetics also supports the education of laboratories. As an enhancement to the Molecular Pathology EQA scheme, a human cell-line reference sample, manufactured by Thermo Fisher Scientific (AcroMetrix), was provided for analysis. This contained many variants, present at frequencies between 1% and 17.9%. METHODS: One hundred and one laboratories submitted results, with a total of 2889 test results on 53 genes being reported. Known polymorphisms, 46/2889 (1.59%) results, were excluded. Variants detected in the seven most commonly reported (and clinically relevant) genes, KRAS, NRAS, BRAF, EGFR, PIK3CA, KIT and PDGFRA, are reported here, as these genes fall within the scope of UK NEQAS EQA schemes. RESULTS: Next generation sequencing (NGS) was the most commonly performed testing platform. There were between 5 and 27 validated variants in the seven genes reported here. Eight laboratories correctly reported all five NRAS variants, and two correctly reported all eight BRAF variants. The validated mean variant frequency was lower than that determined by participating laboratories, with single-gene testing methodologies showing less variation in estimated frequencies than NGS platforms. Laboratories were more likely to correctly identify clinically relevant variants. CONCLUSIONS: Over 100 laboratories took the opportunity to test the 'educational reference sample', showing a willingness to further validate their testing platforms. While it was encouraging to see that the most widely reported variants were those which should be included in routine testing panels, reporting of variants was potentially open to interpretation, thus clarity is still required on whether laboratories selectively reported variants, by either clinical relevance or variant frequency.

Assessment of Capacity to Capture DNA Aerosols by Clean Filters for Molecular Biology Experiments.

Experimental contamination by exogenous DNA is a major issue in molecular biological studies for data quality and its management. We herein assessed DNA aerosols for the risk of contamination and tested the capacity of clean air filters to trap and remove DNA aerosols. DNA aerosols were generated by atomizing a DNA solution and introduced into a laminar flow clean air unit. Capture and detection performed upstream and downstream of the clean air unit showed that a significant fraction (>99.96%) of introduced molecules was trapped and removed by the filter. Although DNA aerosols appear to be an avoidable source of exogenous contamination, a clearer understanding and careful experimental procedures are needed in order to perform contamination-free, high-quality molecular biology experiments.

RAS screening in colorectal cancer: a comprehensive analysis of the results from the UK NEQAS colorectal cancer external quality assurance schemes (2009-2016).

Evidence strongly indicates that extended RAS testing should be undertaken in mCRC patients, prior to prescribing anti-EGFR therapies. With more laboratories implementing testing, the requirement for External Quality Assurance schemes increases, thus ensuring high standards of molecular analysis. Data was analysed from 15 United Kingdom National External Quality Assessment Service (UK NEQAS) for Molecular Genetics Colorectal cancer external quality assurance (EQA) schemes, delivered between 2009 and 2016. Laboratories were provided annually with nine colorectal tumour samples for genotyping. Information on methodology and extent of testing coverage was requested, and scores given for genotyping, interpretation and clerical accuracy. There has been a sixfold increase in laboratory participation (18 in 2009 to 108 in 2016). For RAS genotyping, fewer laboratories now use Roche cobas®, pyrosequencing and Sanger sequencing, with more moving to next generation sequencing (NGS). NGS is the most commonly employed technology for BRAF and PIK3CA mutation screening. KRAS genotyping errors were seen in ≤10% laboratories, until the 2014-2015 scheme, when there was an increase to 16.7%, corresponding to a large increase in scheme participants. NRAS genotyping errors peaked at 25.6% in the first 2015-2016 scheme but subsequently dropped to below 5%. Interpretation and clerical accuracy scores have been consistently good throughout. Within this EQA scheme, we have observed that the quality of molecular analysis for colorectal cancer has continued to improve, despite changes in the required targets, the volume of testing and the technologies employed. It is reassuring to know that laboratories clearly recognise the importance of participating in EQA schemes.

Single-Stranded DNA Library Preparation Preferentially Enriches Short Maternal DNA in Maternal Plasma.

BACKGROUND: Recent studies have suggested that single-stranded DNA (ssDNA) library preparation can enrich short DNA species from the plasma of healthy individuals, cancer patients, and transplant recipients. Based on previous observations that fetal DNA molecules in the maternal plasma are shorter than maternal DNA molecules, ssDNA library preparation may potentially enrich fetal DNA and provide substantial improvement in noninvasive prenatal testing. METHODS: We tested this hypothesis by comparing the maternal plasma DNA sequencing results using 2 types of ssDNA library preparation methods and a standard double-stranded DNA (dsDNA) library method using samples from first- and third-trimester pregnancies. We also evaluated the performance of ssDNA and dsDNA library methods in the noninvasive prenatal detection of trisomy 21 from maternal plasma. RESULTS: Short DNA species were significantly enriched in ssDNA libraries. However, contrary to previous speculation, no significant enrichment was observed in the overall fetal fraction in maternal plasma collected in the first trimester. Our use of an ssDNA library did not reduce the variation in chromosomal representation when compared with a standard dsDNA library in the first-trimester plasma samples. ssDNA libraries also showed inferior performance in the noninvasive prenatal detection of trisomy 21 from maternal plasma. Detailed fetal fraction analysis using size-fractionated Y chromosome sequences and fetal-specific single-nucleotide polymorphisms (SNPs) revealed an unexpected finding that short maternal DNA was preferentially enriched over short fetal DNA in an ssDNA library irrespective of GC content. CONCLUSIONS: Our findings have shown that ssDNA library preparation preferentially enriches short maternally derived DNA in maternal plasma.

Methodological Guidelines for Accurate Detection of Viruses in Wild Plant Species.

Ecological understanding of disease risk, emergence, and dynamics and of the efficacy of control strategies relies heavily on efficient tools for microorganism identification and characterization. Misdetection, such as the misclassification of infected hosts as healthy, can strongly bias estimates of disease prevalence and lead to inaccurate conclusions. In natural plant ecosystems, interest in assessing microbial dynamics is increasing exponentially, but guidelines for detection of microorganisms in wild plants remain limited, particularly so for plant viruses. To address this gap, we explored issues and solutions associated with virus detection by serological and molecular methods in noncrop plant species as applied to the globally important Barley yellow dwarf virus PAV (Luteoviridae), which infects wild native plants as well as crops. With enzyme-linked immunosorbent assays (ELISA), we demonstrate how virus detection in a perennial wild plant species may be much greater in stems than in leaves, although leaves are most commonly sampled, and may also vary among tillers within an individual, thereby highlighting the importance of designing effective sampling strategies. With reverse transcription-PCR (RT-PCR), we demonstrate how inhibitors in tissues of perennial wild hosts can suppress virus detection but can be overcome with methods and products that improve isolation and amplification of nucleic acids. These examples demonstrate the paramount importance of testing and validating survey designs and virus detection methods for noncrop plant communities to ensure accurate ecological surveys and reliable assumptions about virus dynamics in wild hosts.

Epistemology, Ethics, and Progress in Precision Medicine.

The emerging paradigm of precision medicine strives to leverage the tools of molecular biology to prospectively tailor treatments to the individual patient. Fundamental to the success of this movement is the discovery and validation of "predictive biomarkers," which are properties of a patient's biological specimens that can be assayed in advance of therapy to inform the treatment decision. Unfortunately, research into biomarkers and diagnostics for precision medicine has fallen well short of expectations. In this essay, we examine the portfolio of research activities into the excision repair cross complement group 1 (ERCC1) gene as a predictive biomarker for precision lung cancer therapy as a case study in elucidating the epistemological and ethical obstacles to developing new precision medicines.

Recommendations for accreditation of laboratories in molecular biology of hematologic malignancies.

Over recent years, the development of molecular biology techniques has improved the hematological diseases diagnostic and follow-up. Consequently, these techniques are largely used in the biological screening of these diseases; therefore the Hemato-oncology molecular diagnostics laboratories must be actively involved in the accreditation process according the ISO 15189 standard. The French group of molecular biologists (GBMHM) provides requirements for the implementation of quality assurance for the medical molecular laboratories. This guideline states the recommendations for the pre-analytical, analytical (methods validation procedures, quality controls, reagents), and post-analytical conditions. In addition, herein we state a strategy for the internal quality control management. These recommendations will be regularly updated.

GeneGuard: A modular plasmid system designed for biosafety.

Synthetic biology applications in biosensing, bioremediation, and biomining envision the use of engineered microbes beyond a contained laboratory. Deployment of such microbes in the environment raises concerns of unchecked cellular proliferation or unwanted spread of synthetic genes. While antibiotic-resistant plasmids are the most utilized vectors for introducing synthetic genes into bacteria, they are also inherently insecure, acting naturally to propagate DNA from one cell to another. To introduce security into bacterial synthetic biology, we here took on the task of completely reformatting plasmids to be dependent on their intended host strain and inherently disadvantageous for others. Using conditional origins of replication, rich-media compatible auxotrophies, and toxin-antitoxin pairs we constructed a mutually dependent host-plasmid platform, called GeneGuard. In this, replication initiators for the R6K or ColE2-P9 origins are provided in trans by a specified host, whose essential thyA or dapA gene is translocated from a genomic to a plasmid location. This reciprocal arrangement is stable for at least 100 generations without antibiotic selection and is compatible for use in LB medium and soil. Toxin genes ζ or Kid are also employed in an auxiliary manner to make the vector disadvantageous for strains not expressing their antitoxins. These devices, in isolation and in concert, severely reduce unintentional plasmid propagation in E. coli and B. subtilis and do not disrupt the intended E. coli host's growth dynamics. Our GeneGuard system comprises several versions of modular cargo-ready vectors, along with their requisite genomic integration cassettes, and is demonstrated here as an efficient vector for heavy-metal biosensors.

Influence of killing method on Lepidoptera DNA barcode recovery.

The global DNA barcoding initiative has revolutionized the field of biodiversity research. Such large-scale sequencing projects require the collection of large numbers of specimens, which need to be killed and preserved in a way that is both DNA-friendly and which will keep voucher specimens in good condition for later study. Factors such as time since collection, correct storage (exposure to free water and heat) and DNA extraction protocol are known to play a role in the success of downstream molecular applications. Limited data are available on the most efficient, DNA-friendly protocol for killing. In this study, we evaluate the quality of DNA barcode (cytochrome oxidase I) sequences amplified from DNA extracted from specimens collected using three different killing methods (ethyl acetate, cyanide and freezing). Previous studies have suggested that chemicals, such as ethyl acetate and formaldehyde, degraded DNA and as such may not be appropriate for the collection of insects for DNA-based research. All Lepidoptera collected produced DNA barcodes of good quality, and our study found no clear difference in nucleotide signal strength, probability of incorrect base calling and phylogenetic utility among the three different treatment groups. Our findings suggest that ethyl acetate, cyanide and freezing can all be used to collect specimens for DNA analysis.