Methods & Materials in The Gut Microbiome Study by Li et al.

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Table of Contents

Methods
Materials
Reference

Methods

The main method for the study was 16S rRNA sequencing. The method was combined with structural magnetic resonance imaging (sMRI) and functional resting state (rs-fMRI) (Li et al., 2021). The first objective was to investigate differences in fecal microbiota between 38 patients with schizophrenia (SZ). The groups were demographically controlled (NCs). The second goal was to reveal the relation of differences in brain structure and function to each other. Mainly, 76 subjects were considered, including 38 patients from the Affiliated Brain Hospital of Guangzhou Medical University.

The study participants were carefully selected. The bulk of the subjects included patients with stable psychiatric symptoms for >2 weeks. The seven exclusion criteria included current major DSM-IV-TR Axis I diagnoses, somatic illness, history of epilepsy, history of electroconvulsive seizures, breastfeeding, pregnancy, pregnancy planning, alcohol dependence, and lack of legal guardians. The Affiliated Brain Hospital of Guangzhou Medical University approved the study protocol. Each patient provided written informed consent from each subject or their legal guardian. In order to carry out the research, the questionnaires of all the subjects were taken into account to collect general information.

It is relevant in the article that the research protocol was approved by the ethics committee of the Affiliated Brain Hospital of Guangzhou Medical University. All participants had to sign a written informed consent before the research. All patients were interviewed based on general information, including age, gender, BMI, years of education, medication history, and smoking and alcohol use history. Feces were collected from participants who had to fast for 12 hours. The samples were stored at -80 °C until DNA extraction, which is a sound strategy, and all sanitary conditions are followed. In order to isolate DNA, the researchers used 200 mg of each fecal sample. This method of DNA extraction was similar to the protocol described in the previous work of the researchers, which is quite informative.

Materials

The Illumina MiSeq platform proved to be quite efficient for gene rRNA. Sequence data were processed using the QIIME272 application.DADA273 algorithm is used for raw sequencing results, which were demultiplexed and quality controlled. Features with less than two sequences were filtered out. Indicators of alpha and beta diversity were used to structure the microbial community. The use of the Bray-Curtis method revealed the dissimilarity of the beta diversity. Material such as the package from R74 was used to analyze the output matrices. The taxonomic table has undergone normalization due to the relative abundance at different levels of taxa. Two-sample t-tests for MRI indices were applied to compare differences in functional brain activity between SZ patients and NCs.

The researchers efficiently collected MRI data with the help of a high-end Philips Achieva 3 T MRI scanner at the Affiliated Brain Hospital of Guangzhou Medical University. Acquisition of functional data was accomplished through a sequence of echoplanar images (EPI). A T1-weighted 3D gradient-echo sequence was used to image the structure of the brain. Each study participant had to keep their eyes closed, relax but not fall asleep, and move as little as possible. SPM 12 and DPABI were used to process structural data. In this way, “The strategy of warping GM images to the DARTEL template and spatially normalized to Montreal Neurological Institute (MNI) space with modulation was quite clear”(Li et al., 2021). Extraction of the ReHo index was performed thanks to iTe GMV using a specific formula.

In order to more accurately analyze diversity with alpha indices, the researchers chose Faith’s phylogenetic diversity (Faith_PD) and observed species and Shannon’s index. The Kruskal–Wallis H test is used to calculate differences in diversity between groups. Differences in the composition of taxa between samples can be represented in a distance matrix. The vegan package from R74 helped to present the output matrices. Differentiation of indicators was tested using the Mann-Whitney U-test. In this way, “Residual relative abundances of those taxa and MRI indices with significant group differences were calculated to establish the relationship between differential abundance at the genus level and MRI indices” (Li et al., 2021). Controlling age, gender, and years of education using the vglm function in the VGAM75 package played a rather serious role in the findings.

Reference

Li, S., Song, J., Ke, P., Kong, L., Lei, B., Zhou, J., Huang, Y., Li, H., Li, G., Chen, J., Li, X., Xiang, Z., Ning, Y., Wu, F., & Wu, K. (2021). The gut microbiome is associated with brain structure and function in schizophrenia. Scientific Reports, 11, 9743. Web.

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Methods & Materials in The Gut Microbiome Study by Li et al.

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