A practical guide to amplicon and metagenomic analysis of microbiome data

Advances in high-throughput sequencing (HTS) have fostered rapid developments in the field of microbiome research, and massive microbiome datasets are now being generated. However, the diversity of software tools and the complexity of analysis pipelines make it difficult to access this field. Here, we systematically summarize the advantages and limitations of microbiome methods. Then, we recommend specific pipelines for amplicon and metagenomic analyses, and describe commonly-used software and databases, to help researchers select the appropriate tools. Furthermore, we introduce statistical and visualization methods suitable for microbiome analysis, including alpha- and beta-diversity, taxonomic composition, difference comparisons, correlation, networks, machine learning, evolution, source tracing, and common visualization styles to help researchers make informed choices. Finally, a step-by-step reproducible analysis guide is introduced. We hope this review will allow researchers to carry out data analysis more effectively and to quickly select the appropriate tools in order to efficiently mine the biological significance behind the data.

Diagnostic Dilemma of Inflammatory Bowel Disease: Study from a Tertiary Hospital in South India

Introduction: Diagnosis of inflammatory bowel disease (IBD) and other inflammatory conditions of the colon cannot beestablished when only one or few features are present as they share many pathological features. Hence, this study is undertakento develop reproducible criteria which are valid in the diagnosis of IBD and to differentiate it further from ulcerative colitis (UC)and Crohn’s disease (CD).Purpose: The purpose of the study was (1) to study the histopathological patterns of the colonic biopsy specimen, (2) to developthe reproducible criteria which aid in the diagnosis of UC and CD, and (3) to evaluate the extent of interobserver variabilityover the final diagnosis.Materials and Methods: Endoscopic punch biopsy procedure was done for 35 cases with suspected IBD which were sent tothe Histopathology Department of Bengaluru Medical College. Tissue bits were fixed in 10% formalin and processed by theconventional method and embedded in paraffin blocks. Sections from these blocks were stained with hematoxylin and eosinaccording to standard procedures. After initial histomorphological reporting was done, the 35 slides were reported again by twopathologists. Cases with proven malignancy were excluded from the study.Results: UC was diagnosed in 19 cases (53%) of 35 cases followed by indefinite for IBD in nine cases (25%). CD was seenin five cases (14%) followed by tuberculosis in two cases (5%). One case had evidence of dysplasia along with features ofUC. The agreement between pathologists for the final diagnosis is 68.5%. Based on P value, the significant features whichare most useful in the diagnosis of UC are: For activity – (1) cryptitis, (2) neutrophilic infiltration in lamina propria, and (3) cryptabscess; for chronicity – (1) crypt distortion, (2) crypt branching, (3) Mucin depletion, (4) crypt atrophy, and (5) crypt dilation.Conclusion: UC was found to be more commonly reported among the IBD cases. Considerable disagreement can be seenbetween experienced pathologists reporting the same slides. Therefore, salient histological features based on better reproducibilityalong with adequate clinical and endoscopy findings can aid in distinguishing between UC and CD.

UV Spectrophotometric Determination Of Rupatadine Fumarate In Bulk And Tablet Dosage Form By Using Single Point Standardization Method

Objective: To develop and validate a simple, selective, precise and accurate method for the estimation of rupatadine fumarate in bulk and tablet dosage form by using the single point standardization method as per international conference on harmonization (ICH) guidelines. Methods: In this proposed method, the absorbance of a standard solution of known concentration and a sample solution was measured. From this, the concentration of the unknown can be calculated. Results: Rupatadine fumarate showed maximum absorbance at 246 nm with methanol. Linearity was checked in different concentrations. The calibration curve was obtained in the range of 2-10 µg/ml. The slope, intercept and correlation coefficient (R2) values of Rupatadine fumarate were found to be 0.047, 0.0034 and 0.9995 respectively. Intra-day and inter-day precision studies were carried out and there % RSD values were found within limits i.e. less than 2%. The recovery studies were carried out by adding a known amount of standard drug to preanalysed formulation and % Recovery was found to be within 99.7-101.6%. LOD and LOQ of Rupatadine fumarate were found to be 0.1 µg/ml and 0.3 µg/ml respectively. Robustness studies were performed at different wavelengths and the % RSD was found within the limits i.e. less than 2 %. Conclusion: The developed single point standardization method for the estimation of Rupatadine fumarate was found to be simple, precise, accurate, reproducible and cost-effective. Statistical analysis of the developed method confirms that the proposed method is an appropriate and it can be useful for the routine analysis. The proposed method gives the basic idea to the researcher who is working in the area like product development.

Investigación cualitativa reproducible: un camino que debemos empezar a recorrer / Qualitative reproducible research: A path we must begin to walk

El movimiento conocido como investigación reproducible (IR) ha venido ganando espacio dentro de la investigación cuantitativa como una forma de aumentar la confianza en sus resultados. El movimiento propone que los datos primarios y la codificación usada en el análisis de una investigación estén disponibles para que otros investigadores puedan verificar los resultados publicados y conducir análisis alternativos. Las ventajas asociadas a la IR aplican muy bien dentro del trabajo de los investigadores cualitativos, quienes no solo gozarían de mayor confianza y aceptación de los resultados de su investigación, sino que podrían beneficiarse al permitir que las personas interesadas entiendan sus hallazgos e, incluso, al favorecer la formación de redes de investigación y la construcción de conocimiento de forma colaborativa. Las limitaciones más grandes para que se pueda realizar investigación cualitativa (IC) reproducible están dadas por las restricciones éticas intrínsecas a hacer disponible de forma abierta los datos primarios y la codificación de los estudios. En el presente artículo se presentan algunas propuestas para superar estas dificultades, en lo referente al consentimiento informado y medidas para garantizar la confidencialidad de los datos, reconociendo que aún falta mucho camino superar estas dificultades. Así mismo, se presentan algunos pasos que ya se han avanzado para permitir que se desarrolle IC reproducible incluyendo el desarrollo de programas informáticos para el manejo de datos cualitativos, la disponibilidad de repositorios para hacer disponibles esos datos y los avances que se han desarrollado en la implementación de IR cuantitativa.

The movement known as reproducible research (RR) has won space in the quantitative research field as a way to increase the confidence in their results. The RR movement proposes that primary data and Computer codes used in the research must be publically available to let other researchers verify published results and to conduct alternative analysis. The advantages related to RR apply well to the qualitative researcher's work, and will increase the confidence and approval of their results, and additionally could provide advantages like the deeper understanding of their findings, the chance to create investigation networks and groups for collaborative knowledge building. The biggest limitations to produce qualitative reproducible research are ethical considerations associated with making sensitive information openlv available. The present article presents some proposals to overeóme these difficulcies associated with the process of obtaining informed consent and measures to guarantee the confidentiality of data, recognizing that there is much work to be done in this area. Additionally, some steps have been taken to facilitare reproducible qualitative research, including the development of software packages to manage qualitative data, the availability of repositories to make data available and advances developed in reproducible quantitative research implementation.

Simultaneous determination of chloramphenicol and hydrocortisone acetate in cream using TLC desitrometry method.

A rapid and reproducible TLC method was developed for the determination of hydrocortisone acetate and chloram-phenicol in cream. The analytes were dissolved with methanol and chromatographed on silica Gel GF 254 TLC plate using chloroform:ethyl acetate in the ratio of 1:1.5 (v/v) as mobile phase. Spots at Rf 0.29 and Rf 0.59 were recognized as chloramphenicol and hydrocortisone acetate, respectively. Quantitative analysis was done through densitometric measurement at wavelength 265 nm. Method was found linear over the concentration range of 300-900 ng/spot with the correlation coefficient of 0.999 and 0.998 for hydrocortisone acetate and chloramphenicol, respectively. Specificity showed calculation of purity and identity more than 0.99. The limit of detection (LOD) and the limit of quantification (LOQ) of the method were 23.84 and 71.51 ng/spot for hydrocortisone acetate, 21.06 and 63.18 ng/spot for chloram-phenicol. The precision of this method was less than 2.8% whereas the means of the recovery data were 100.40± 0.579% for hydrocortisone acetate and 100.24±1.20% for chloramphenicol. The proposed method has been applied to the determination of hydrocortisone acetate and chloramphenicol in commercial cream formulations and the recovery of label claim were 99.23±0.66% (chloramphenicol) and 99.25±0.41% (hydrocortisone acetate) for brand A and 100.32±0.87% (chloramphenicol) and 100.53±0.78% (hydrocortisone acetate) for brand B. The developed method was successfully used for the assay of hydrocortisone acetate and chloramphenicol. The method is simple, sensitive and precise; it can be used for the routine quality control testing of marketed formulations.

Simultaneous determination of chloramphenicol and hydrocortisone acetate in cream using TLC desitrometry method.

A rapid and reproducible TLC method was developed for the determination of hydrocortisone acetate and chloram-phenicol in cream. The analytes were dissolved with methanol and chromatographed on silica Gel GF 254 TLC plate using chloroform:ethyl acetate in the ratio of 1:1.5 (v/v) as mobile phase. Spots at Rf 0.29 and Rf 0.59 were recognized as chloramphenicol and hydrocortisone acetate, respectively. Quantitative analysis was done through densitometric measurement at wavelength 265 nm. Method was found linear over the concentration range of 300-900 ng/spot with the correlation coefficient of 0.999 and 0.998 for hydrocortisone acetate and chloramphenicol, respectively. Specificity showed calculation of purity and identity more than 0.99. The limit of detection (LOD) and the limit of quantification (LOQ) of the method were 23.84 and 71.51 ng/spot for hydrocortisone acetate, 21.06 and 63.18 ng/spot for chloram-phenicol. The precision of this method was less than 2.8% whereas the means of the recovery data were 100.40± 0.579% for hydrocortisone acetate and 100.24±1.20% for chloramphenicol. The proposed method has been applied to the determination of hydrocortisone acetate and chloramphenicol in commercial cream formulations and the recovery of label claim were 99.23±0.66% (chloramphenicol) and 99.25±0.41% (hydrocortisone acetate) for brand A and 100.32±0.87% (chloramphenicol) and 100.53±0.78% (hydrocortisone acetate) for brand B. The developed method was successfully used for the assay of hydrocortisone acetate and chloramphenicol. The method is simple, sensitive and precise; it can be used for the routine quality control testing of marketed formulations.

Controle de qualidade intralaboratorial em hematologia: avaliação da precisão e exatidão dos equipamentos semi-automatizados (contador de leucócitos, hemoglobinômetro, microhematócrito, diluidor automático), no "Instituto Lauro de Souza Lima"

Neste estudo investigou-se o grau de reprodutividade e sensibilidade dos aparelhos hematológicos (Contador de leucócitos, hemoglobinômetro, microhematócrito), utilizados no laboratório clínico do Instituto Lauro de Souza Lima de Bauru, usando-se para isto 50 amostras triadas apenas de pacientes ambulatorial, da rotina do mesmo laboratório. Os resultados obtidos demonstram que independente do tempo de uso dos aparelhos, desde que se faça uma manutenção periódica nos mesmos, pode-se obter resultados confiáveis.