Application Of 16S And Metagenomic Sequencing Technologies In Food Microbiology And Gut Microbiology

The early days of microbial research relied on artificial culture,but for most microorganisms,the conditions and environment of artificial culture are not available,which has prevented researchers from studying and understanding microorganisms in depth.With the rapid development of high-throughput sequencing technology in recent years,research on microorganisms that cannot be cultured artificially has become clearer.In the early days,microorganisms were studied by one-generation sequencing of microorganisms that could be cultured artificially,but not by those that could not be cultured artificially.This paper will be a study of microorganisms that are relevant to everyday life.In this paper,we use 16S rRNA sequencing and macro-genome sequencing to study the microbial community structure and species diversity of food microorganisms and human gut microorganisms,which are closely related to daily life,and establish a standardised experimental procedure and bioinformatic analysis process.In this paper,we have done the following research work.Firstly,this chapter focuses on the Pixian douban microorganism,which consists of four different fermentation stages: the dried chilli fermentation stage,the broad bean douban fermentation stage,the red oil douban fermentation stage and the Pixian douban fermentation stage.The research method was to use 16S rRNA sequencing technology to first extract the metagenomic DNA and then apply fluorescent quantitative PCR to detect the bacterial count in the samples.This study was followed by the construction of a high-throughput sequencing library of the V3V4 region of the 16S rRNA gene using two amplification methods,which was sequenced using a sequencing platform.A high-throughput data manipulation process for direct comparison of short sequences was then established for the analysis of microbial species diversity in this type of study.In this paper,the sequences obtained from sequencing were firstly spliced into a target region sequence using FLASH,and then compared with the reference gene library to obtain the target sequence.Then the species taxonomy was annotated based on the optimized clustering algorithm,which improved the efficiency of sequence utilization.Bioinformatics analysis was performed on the annotated OTUs,which included Alpha diversity analysis,Beta diversity analysis,differential flora analysis of different Pixian douban fermentation stages and functional prediction analysis of Pixian douban flora.In the Alpha diversity analysis,the Shannon index and Chao1 index were compared to analyse the differences in bacterial diversity and richness,and the Beta diversity analysis mainly analysed the similarities and differences of bacterial flora at different fermentation stages by principal components.The results showed that the bacterial community of Pixian douban was in dynamic stability during the initial fermentation process,and the bacterial community composition was relatively rich at different fermentation stages;from the whole initial fermentation process of Pixian douban,the bacterial community diversity showed an increasing trend,and the Shannon Shannon index increased from 1.25 to 3.5;the number and diversity of bacterial community in the initial fermentation process of Pixian douban were closely related to the fermentation The number and diversity of bacterial communities in the initial fermentation of Pixian douban were closely related to the fermentation environment,and the diversity of bacterial communities varied at different stages of fermentation,with Pantoea being the most dominant genus in the dry chilli fermentation stage,accounting for20%;Staphylococcus had the highest relative abundance in the broad bean flap fermentation stage,accounting for 38%;after mixed fermentation,the most dominant genus in the red oil douban fermentation stage was Lactobacillus.After mixed fermentation,the most dominant genus in the fermentation stage of red bean paste was Lactobacillus,accounting for 27%,and the most dominant genus in the fermentation stage of Pixian bean paste was Lactobacillus,accounting for 62%.Secondly,the study of human gut microbes is focused on healthy populations,diabetic and glycocentric patients.The study uses Pac Bio’s CCS(Circular Consensus Sequencing)sequencing model,which combines long read length with high accuracy.The ultra-deep sequencing allows for more genomic data and features than conventional macro-genomic sequencing of 5Gbp and 10 Gbp,which greatly improves the data richness and processing efficiency.In this study,a total of 2,019 drug resistance genes were identified in 24 tri-generation macro-genomic sequencing data,with approximately 87 species resistant to 14 antibiotics,including macrolide,tetracycline,aminoglycoside and tetracycline.aminoglycoside)resistance genes.There were 8770 mobile elements,including integrated splice elements(ICE),plasmid,prophage,integron and insertion sequence(IS),with the largest number of ICE.The MAGs were then annotated with resistance genes after binning and were found to carry a total of 765 resistance genes,which were classified into 70 species according to ARG type,mainly tetracycline resistance genes,followed by macrolide and aminoglycoside resistance genes.An in-depth analysis of the types of resistance genes carried by different species can lead to the conclusion that mobile elements in human gut microbes carry resistance genes for gene level transfer,which also provides a certain degree of help in understanding and treating antibiotic resistance.