Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.

YeastFab Cloning of Toxic Genes and Protein Expression Optimization in Yeast.

YeastFab is a Golden Gate-based cloning standard and parts repository. It is designed for modular, hierarchical assembly of transcription units and multi-gene assemblies for expression in Saccharomyces cerevisiae. This makes it a suitable toolbox to optimize the expression strength of heterologous genes in yeast. When cloning heterologous coding sequences into YeastFab vectors, in several cases we have observed toxicity to the cloning host Escherichia coli. The provided protocol details how to clone such toxic genes from multiple synthetic DNA fragments while adhering to the YeastFab standard. The presented cloning strategy includes a C-terminal FLAG tag that allows screening for constructs with a desired protein expression in yeast by western blot. The design allows scarlessly removing the tag through a Golden Gate reaction to facilitate cloning of expression constructs with the native, untagged transgene.

Targeted Photoactivatable Green-Emitting BODIPY Based on Directed Photooxidation-Induced Activation and its Application to Live Dynamic Super-Resolution Microscopy.

Photoactivatable fluorescent probes are valuable tools in bioimaging for tracking cells down to single molecules and for single molecule localization microscopy. For the latter application, green emitting dyes are in demand. We herein developed an efficient green-emitting photoactivatable furanyl-BODIPY (PFB) and we established a new mechanism of photoactivation called Directed Photooxidation Induced Activation (DPIA) where the furan is photo-oxidized in a directed manner by the singlet oxygen produced by the probe. The efficient photoconverter (93-fold fluorescence enhancement at 510 nm, 49% yield conversion) is functionalizable and allowed targeting of several subcellular structures and organelles, which were photoactivated in live cells. Finally, we demonstrated the potential of PFB in super-resolution imaging by performing PhotoActivated Localization Microscopy (PALM) in live cells.

Passage efficiency through fishways of species of the family Cyprinidae and their management implications for fragmented rivers.

The contemporary management of fragmented river systems is in a trade-off between the societal benefits of instream barriers (e.g. hydropower, flood risk management) and the ecological harms of their adverse impacts on fish populations. The consequent fragmentation can be mitigated through fishway construction, with mitigation performance measured using species-specific passage rates and efficiencies. There is, however, a bias in passage efficiency studies towards diadromous fishes and, although fish of the Cyprinidae family play a significant role in the fish assemblages of rivers worldwide, their passage efficiencies are poorly understood. Here, systematic review and meta-analyses assessed the passage efficiencies of cyprinid fishes through fishways that have been measured using telemetry methods. Passive integrated transponder (PIT) telemetry was the most common evaluation method of passage efficiency due to their high read rates and relatively low costs versus alternative telemetry methods. These methods revealed cyprinid passage efficiencies were highest through vertical slot fishways and lowest through nature-like constructions, with overall passage rates comparing favourably to anadromous salmonid fishes. Fish were most active during spring and summer, with passage and associated movements often related to spawning. Passage rates of non-native fishes were also higher than for native fishes. Despite the growing acknowledgment of how fishways influence potamodromous fish dispersal and distribution in rivers, passage data remain scarce, preventing managers and policy-makers from making informed decisions on optimal passage solutions for multiple fish species in highly fragmented rivers.

The concentration of free glycerol in goat milk increases during feed restrictions.

This Research Communication introduces a novel enzymatic-fluorometric analytical procedure for glycerol and glycerol 3-phosphate in milk. Milk from thirty-seven goats was analysed during 9 consecutive days during which a two-day feed restriction was introduced. Fractional milk triacylglyceride and free glycerol increased significantly while glycerol 3-phosphate reacted more moderately. The energy status of the mammary cell is discussed.

Magnetic microbead-based upconversion immunoassay with laser-induced breakdown spectroscopy readout for the detection of prostate-specific antigen.

Laser-induced breakdown spectroscopy (LIBS) is a promising technique for the readout of immunochemical assays utilizing indirect detection of labels (Tag-LIBS), typically based on nanoparticles. We have previously demonstrated that Tag-LIBS immunoassay employing yttrium-based photon-upconversion nanoparticles (UCNPs) can reach sensitivity similar to commonly used enzyme and fluorescence immunoassays. In this study, we report on further increasing the sensitivity of UCNP-based Tag-LIBS immunoassay by employing magnetic microbeads (MBs) as the solid phase in the determination of cancer biomarker prostate-specific antigen. Due to the possibility of analyte preconcentration, MBs enabled achieving a limit of detection (LOD) of 4.0 pg·mL-1, representing two orders of magnitude improvement compared with equivalent microtiter plate-based assay (LOD of 460 pg·mL-1). In addition, utilizing MBs opens up the possibility of an internal standardization of the LIBS readout by employing iron spectral lines, which improves the assay robustness by compensating for LIBS signal fluctuations and bead-bound immunocomplexes lost throughout the washing steps. Finally, the practical applicability of the technique was confirmed by the successful analysis of clinical samples, showing a strong correlation with the standard electrochemiluminescence immunoassay. Overall, MB-based Tag-LIBS was confirmed as a promising immunoassay approach, combining fast readout, multiplexing possibilities, and high sensitivity approaching upconversion luminescence scanning while avoiding the requirement of luminescence properties of labels.

Enhancer looping protein LDB1 modulates MYB expression in T-ALL cell lines in vitro by cooperating with master transcription factors.

BACKGROUND: Despite significant progress in the prognosis of pediatric T-cell acute lymphoblastic leukemia (T-ALL) in recent decades, a notable portion of children still confronts challenges such as treatment resistance and recurrence, leading to limited options and a poor prognosis. LIM domain-binding protein 1 (LDB1) has been confirmed to exert a crucial role in various physiological and pathological processes. In our research, we aim to elucidate the underlying function and mechanisms of LDB1 within the background of T-ALL. METHODS: Employing short hairpin RNA (shRNA) techniques, we delineated the functional impact of LDB1 in T-ALL cell lines. Through the application of RNA-Seq, CUT&Tag, and immunoprecipitation assays, we scrutinized master transcription factors cooperating with LDB1 and identified downstream targets under LDB1 regulation. RESULTS: LDB1 emerges as a critical transcription factor co-activator in cell lines derived from T-ALL. It primarily collaborates with master transcription factors (ERG, ETV6, IRF1) to cooperatively regulate the transcription of downstream target genes. Both in vitro and in vivo experiments affirm the essential fuction of LDB1 in the proliferation and survival of cell lines derived from T-ALL, with MYB identified as a significant downstream target of LDB1. CONCLUSIONS: To sum up, our research establishes the pivotal fuction of LDB1 in the tumorigenesis and progression of T-ALL cell lines. Mechanistic insights reveal that LDB1 cooperates with ERG, ETV6, and IRF1 to modulate the expression of downstream effector genes. Furthermore, LDB1 controls MYB through remote enhancer modulation, providing valuable mechanistic insights into its involvement in the progression of T-ALL.

Targeting TAG-72 in cutaneous T cell lymphoma.

Purpose: Current monoclonal antibody-based treatment approaches for cutaneous T cell lymphoma (CTCL) rely heavily on the ability to identify a tumor specific target that is essentially absent on normal cells. Herein, we propose tumor associated glycoprotein-72 (TAG-72) as one such target. TAG-72 is a mucin-associated, truncated O-glycan that has been identified as a chimeric antigen receptor (CAR)-T cell target in solid tumor indications. To date, TAG-72 targeting has not been considered in the setting of hematological malignancies. Experimental design: CD3+ cells from patients with CTCL were analyzed for TAG-72 expression by flow cytometry. Immunohistochemistry was used to assess TAG-72 expression in CTCL patient skin lesions and a TAG-72 ELISA was employed to assess soluble TAG-72 (CA 72-4) in patient plasma. TAG-72 CAR transduction was performed on healthy donor (HD) and CTCL T cells and characterized by flow cytometry. In vitro CAR-T cell function was assessed by flow cytometry and xCELLigence® using patient peripheral blood mononuclear cells and proof-of-concept ovarian cancer cell lines. In vivo CAR-T cell function was assessed in a proof-of-concept, TAG-72+ ovarian cancer xenograft mouse model. Results: TAG-72 expression was significantly higher on total CD3+ T cells and CD4+ subsets in CTCL donors across disease stages, compared to that of HDs. TAG-72 was also present in CTCL patient skin lesions, whereas CA 72-4 was detected at low levels in both CTCL patient and HD plasma with no differences between the two groups. In vitro cytotoxicity assays showed that anti-TAG-72 CAR-T cells significantly, and specifically reduced CD3+TAG-72+ expressing CTCL cells, compared to culture with unedited T cells (no CAR). CTCL CAR-T cells had comparable function to HD CAR-T cells in vitro and CAR-T cells derived from CTCL patients eradicated cancer cells in vivo. Conclusion: This study shows the first evidence of TAG-72 as a possible target for the treatment of CTCL.

Engineering of heterobifunctional biopolymers for tunable binding and precipitation of Strep-Tag proteins and virus-like nanoparticles.

Affinity precipitation is a powerful separation method in that it combines the binding selectivity of affinity chromatography with precipitation of captured biomolecules via phase separation triggered by small changes in the environment, e.g., pH, ionic strength, temperature, light, etc. Elastin-like polypeptides (ELPs) are thermally responsive biopolymers composed of pentapeptide repeats VPGVG that undergo reversible phase separation, where they aggregate when temperature and/or salt concentration are increased. Here we describe the generation of an ELP fusion to a soluble streptavidin mutant that enables rapid purification of any Strep-tag II fusion protein of interest. This heterobifunctional protein takes advantage of the native tetrameric structure of streptavidin, leading to binding-induced multivalent crosslinking upon protein capture. The efficient biotin-mediated dissociation of the bound Strep-tag II fusion protein from the streptavidin-ELP capturing scaffold allows for mild elution conditions. We also show that this platform is particularly effective in the purification of a virus-like particle (VLP)-like E2 protein nanoparticle, likely because the high valency of the protein particle causes binding-induced crosslinking and precipitation. Considering the importance of VLP for gene therapy applications, we believe this is a particularly exciting advance. We demonstrated this feasibility by the efficient purification of a VLP-like E2 protein nanoparticle as a surrogate.

A Study of Small Intestinal Epigenomic Changes Induced by Royal Jelly.

This study explores the impact of royal jelly (RJ) on small intestinal epigenomic changes. RJ, produced by honeybees, is known for its effects on metabolic diseases. The hypothesis is that RJ induces epigenomic modifications in small intestinal epithelial cells, affecting gene expression and contributing to metabolic health. Male db/m and db/db mice were used to examine RJ's effects through mRNA sequencing and CUT&Tag methods. This study focused on histone modifications and gene expression changes, with statistical significance set at p < 0.05. RJ administration improved insulin sensitivity and lipid metabolism without affecting body weight. GO and KEGG pathway analyses showed significant enrichment in metabolic processes, cellular components, and molecular functions. RJ altered histone modifications, increasing H3K27me3 and decreasing H3K23Ac in genes associated with the G2M checkpoint. These genes, including Smc2, Mcm3, Ccnd1, Rasal2, Mcm6, and Mad2l1, are linked to cancer progression and metabolic regulation. RJ induces beneficial epigenomic changes in small intestinal epithelial cells, improving metabolic health and reducing cancer-associated gene expression. These findings highlight RJ's potential as a therapeutic agent for metabolic disorders. Further research is needed to fully understand the mechanisms behind these effects and their implications for human health.

In-situ nanozyme catalytic amplification coupled with a universal antibody orientation strategy based electrochemical immunosensor for AD-related biomarker.

An in-situ nanozyme signal tag combined with a DNA-mediated universal antibody-oriented strategy was proposed to establish a high-performance immunosensing platform for Alzheimer's disease (AD)-related biomarker detection. Briefly, a Zr-based metal-organic framework (MOF) with peroxidase (POD)-like activity was synthesized to encapsulating the electroactive molecule methylene blue (MB), and subsequently modified with a layer of gold nanoparticles on its surface. This led to the creation of double POD-like activity nanozymes surrounding the MB molecule to form a nanozyme signal tag. A large number of hydroxyl radicals were generated by the nanozyme signal tag with the help of H2O2, which catalyzed MB molecules in situ to achieve efficient signal amplification. Subsequently, a DNA-aptamer-mediated universal antibody-oriented strategy was proposed to enhance the binding efficiency for the antigen (target). Meanwhile, a poly adenine was incorporated at the end of the aptamer, facilitating binding to the gold electrode and providing anti-fouling properties due to the hydrophilicity of the phosphate group. Under optimal conditions, this platform was successfully employed for highly sensitive detection of AD-associated tau protein and BACE1, achieving limits of detection with concentrations of 3.34 fg/mL and 1.67 fg/mL, respectively. It is worth mentioning that in the tau immunosensing mode, 20 clinical samples from volunteers of varying ages were analyzed, revealing significantly higher tau expression levels in the blood samples of elderly volunteers compared to young volunteers. This suggests that the developed strategy holds great promise for early AD diagnosis.

Seasonal habitat use and diel vertical migration in female spurdog in Nordic waters.

BACKGROUND: Studying habitat use and vertical movement patterns of individual fish over continuous time and space is innately challenging and has therefore largely remained elusive for a wide range of species. Amongst sharks, this applies particularly to smaller-bodied and less wide-ranging species such as the spurdog (Squalus acanthias Linnaeus, 1758), which, despite its importance for fisheries, has received limited attention in biologging and biotelemetry studies, particularly in the North-East Atlantic. METHODS: To investigate seasonal variations in fine-scale niche use and vertical movement patterns in female spurdog, we used archival data from 19 pregnant individuals that were satellite-tagged for up to 365 days in Norwegian fjords. We estimated the realised niche space with kernel densities and performed continuous wavelet analyses to identify dominant periods in vertical movement. Triaxial acceleration data were used to identify burst events and infer activity patterns. RESULTS: Pregnant females frequently utilised shallow depths down to 300 m at temperatures between 8 and 14 °C. Oscillatory vertical moments revealed persistent diel vertical migration (DVM) patterns, with descents at dawn and ascents at dusk. This strict normal DVM behaviour dominated in winter and spring and was associated with higher levels of activity bursts, while in summer and autumn sharks predominantly selected warm waters above the thermocline with only sporadic dive and bursts events. CONCLUSIONS: The prevalence of normal DVM behaviour in winter months linked with elevated likely foraging-related activity bursts suggests this movement behaviour to be foraging-driven. With lower number of fast starts exhibited in warm waters during the summer and autumn months, habitat use in this season might be rather driven by behavioural thermoregulation, yet other factors may also play a role. Individual and cohort-related variations indicate a complex interplay of movement behaviour and habitat use with the abiotic and biotic environment. Together with ongoing work investigating fine-scale horizontal movement as well as sex- and age-specific differences, this study provides vital information to direct the spatio-temporal distribution of a newly reopened fishery and contributes to an elevated understanding of the movement ecology of spurdog in the North-East Atlantic and beyond.

A unified purification method for actin-binding proteins using a TEV-cleavable His-Strep-tag.

The actin cytoskeleton governs the dynamic functions of cells, ranging from motility to phagocytosis and cell division. To elucidate the molecular mechanism, in vitro reconstructions of the actin cytoskeleton and its force generation process have played essential roles, highlighting the importance of efficient purification methods for actin-binding proteins. In this study, we introduce a unified purification method for actin-binding proteins, including capping protein (CP), cofilin, ADF, profilin, fascin, and VASP, key regulators in force generation of the actin cytoskeleton. Exploiting a His-Strep-tag combined with a TEV protease cleavage site, we purified these diverse actin-binding proteins through a simple two-column purification process: initial purification through a Strep-Tactin column and subsequent tag removal through the reverse purification by a Ni-NTA column. Biochemical and microscopic assays validated the functionality of the purified proteins, demonstrating the versatility of the approach. Our methods not only delineate critical steps for the efficient preparation of actin-binding proteins but also hold the potential to advance investigations of mutants, isoforms, various source species, and engineered proteins involved in actin cytoskeletal dynamics.•Unified purification method for various actin-binding proteins.•His-Strep-tag and TEV protease cleavage for efficient purification.•Functional validation through biochemical and microscopic assays.

Genetic Diversity of Tulipa alberti and T. greigii Populations from Kazakhstan Based on Application of Expressed Sequence Tag Simple Sequence Repeat Markers.

The genus Tulipa L., renowned for its ornamental and ecological significance, encompasses a diversity of species primarily concentrated in the Tian Shan and Pamir-Alay Mountain ranges. With its varied landscapes, Kazakhstan harbors 42 Tulipa species, including the endangered Tulipa alberti Regel and Tulipa greigii Regel, which are critical for biodiversity yet face significant threats from human activities. This study aimed to assess these two species' genetic diversity and population structure using 15 expressed sequence tag simple sequence repeat (EST-SSR) markers. Leaf samples from 423 individuals across 23 natural populations, including 11 populations of T. alberti and 12 populations of T. greigii, were collected and genetically characterized using EST-SSR markers. The results revealed relatively high levels of genetic variation in T. greigii compared to T. alberti. The average number of alleles per locus was 1.9 for T. alberti and 2.8 for T. greigii. AMOVA indicated substantial genetic variation within populations (75% for T. alberti and 77% for T. greigii). The Bayesian analysis of the population structure of the two species indicated an optimal value of K = 3 for both species, splitting all sampled populations into three distinct genetic clusters. Populations with the highest level of genetic diversity were identified in both species. The results underscore the importance of conserving the genetic diversity of Tulipa populations, which can help develop strategies for their preservation in stressed ecological conditions.

Preclinical lentiviral hematopoietic stem cell gene therapy corrects Pompe disease-related muscle and neurological manifestations.

Pompe disease, a rare genetic neuromuscular disorder, is caused by a deficiency of acid alpha-glucosidase (GAA), leading to an accumulation of glycogen in lysosomes, and resulting in the progressive development of muscle weakness. The current standard treatment, enzyme replacement therapy (ERT), is not curative and has limitations such as poor penetration into skeletal muscle and both the central and peripheral nervous systems, a risk of immune responses against the recombinant enzyme, and the requirement for high doses and frequent infusions. To overcome these limitations, lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy has been proposed as a next-generation approach for treating Pompe disease. This study demonstrates the potential of lentiviral HSPC gene therapy to reverse the pathological effects of Pompe disease in a preclinical mouse model. It includes a comprehensive safety assessment via integration site analysis, along with single-cell RNA sequencing analysis of central nervous tissue samples to gain insights into the underlying mechanisms of phenotype correction.

An assessment of seasonal bait uptake by individual grey squirrels to develop a delivery system for oral contraceptives.

Globally, human-wildlife conflicts continue to increase, owing to human population growth and expansion. Many of these conflicts concern the impacts of invasive non-native species. In the UK, the invasive, non-native grey squirrel Sciurus carolinensis negatively affects tree health and has caused the decline of the native red squirrel Sciurus vulgaris. Oral contraceptives are being developed to manage the impacts of the grey squirrel. To be effective, contraceptives will need to be deployed at a landscape scale, and will require a delivery system that is practical and economically viable. Understanding grey squirrel feeding behaviour is important so that delivery methods can be designed so that a sufficient number of target individuals receive an effective contraceptive dose at a time of year that will ensure their infertility throughout peak times of breeding. The main aims of this study were to assess how sex, season, squirrel density and bait point density influenced; (1) the probability of a squirrel visiting a feeder and (2) the amount of bait consumed from feeders. Field trials were conducted on six woodland populations of squirrels in three seasons, with four days of bait deployment via purpose-designed squirrel-specific bait hoppers with integrated PIT-tag readers. It was possible to deliver multiple doses on most days to most male and female grey squirrels, with bait deployment more likely to be effective in spring, immediately before the second annual peak in squirrel breeding, followed by winter, immediately before the first peak in breeding. The results from this study could be used to design methods for delivering oral contraceptive baits to grey squirrels in the future and the methods used could be applied to other small mammal species and other bait delivery systems. © 2024 Crown copyright and The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. This article is published with the permission of the Controller of HMSO and the King's Printer for Scotland.

Contrastive Learning-Based Personalized Tag Recommendation.

Personalized tag recommendation algorithms generate personalized tag lists for users by learning the tagging preferences of users. Traditional personalized tag recommendation systems are limited by the problem of data sparsity, making the personalized tag recommendation models unable to accurately learn the embeddings of users, items, and tags. To address this issue, we propose a contrastive learning-based personalized tag recommendation algorithm, namely CLPTR. Specifically, CLPTR generates augmented views of user-tag and item-tag interaction graphs by injecting noises into implicit feature representations rather than dropping nodes and edges. Hence, CLPTR is able to greatly preserve the underlying semantics of the original user-tag or the item-tag interaction graphs and avoid destroying their structural information. In addition, we integrate the contrastive learning module into a graph neural network-based personalized tag recommendation model, which enables the model to extract self-supervised signals from user-tag and item-tag interaction graphs. We conduct extensive experiments on real-world datasets, and the experimental results demonstrate the state-of-the-art performance of our proposed CLPTR compared with traditional personalized tag recommendation models.

Plasmonic Nanotrenches with 1 nm Nanogaps for Surface-Enhanced Raman Scattering-Based Screening of His-Tagged Proteins.

This study represents a highly sensitive and selective approach to protein screening using surface-enhanced Raman scattering (SERS) facilitated by octahedral Au nanotrenches (OANTs). OANTs are a novel class of nanoparticles characterized by narrow, trench-like excavations indented into the eight facets of a Au octahedron. This unique configuration maximizes electromagnetic near-field focusing as the gap distance decreases to ∼1 nm. Owing to geometrical characteristics of the OANTs, near-field focusing can be maximized through the confinement and reflectance of light trapped within the trenches. We used Ni ions and molecular linkers to confer selective binding affinity for His-tagged proteins on the surfaces of the OANTs for SERS-based protein screening. Remarkably, SERS-based protein screening with the surface-modified OANTs yielded outstanding screening capabilities: 100% sensitivity and 100% selectivity in distinguishing His-tagged human serum albumin (HSA) from native HSA. This highlights the significantly enhanced protein screening capabilities achieved through the synergistic combination of SERS and the OANTs.

Review and Evaluate the Bioinformatics Analysis Strategies of ATAC-seq and CUT&Tag Data.

Efficient and reliable profiling methods are essential to study epigenetics. Tn5, one of the first identified prokaryotic transposases with high DNA-binding and tagmentation efficiency, is widely adopted in different genomic and epigenomic protocols for high-throughputly exploring the genome and epigenome. Based on Tn5, the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and the Cleavage Under Targets and Tagmentation (CUT&Tag) were developed to measure chromatin accessibility and detect DNA-protein interactions. These methodologies can be applied to large amounts of biological samples with low-input levels, such as rare tissues, embryos, and sorted single cells. However, fast and proper processing of these epigenomic data has become a bottleneck because massive data production continues to increase quickly. Furthermore, inappropriate data analysis can generate biased or misleading conclusions. Therefore, it is essential to evaluate the performance of Tn5-based ATAC-seq and CUT&Tag data processing bioinformatics tools, many of which were developed mostly for analyzing chromatin immunoprecipitation followed by sequencing (ChIP-seq) data. Here, we conducted a comprehensive benchmarking analysis to evaluate the performance of eight popular software for processing ATAC-seq and CUT&Tag data. We compared the sensitivity, specificity, and peak width distribution for both narrow-type and broad-type peak calling. We also tested the influence of the availability of control IgG input in CUT&Tag data analysis. Finally, we evaluated the differential analysis strategies commonly used for analyzing the CUT&Tag data. Our study provided comprehensive guidance for selecting bioinformatics tools and recommended analysis strategies, which were implemented into Docker/Singularity images for streamlined data analysis.