A Study On Functional Genomics Of Bacterial Flora Associated With Bacterial Vaginosis

ObjectiveThis project intends to establish a bacterial molecular biology technique that does not rely on culture,to obtain bacterial genome-wide DNA samples of normal and BV women's vaginal micro-ecological system environments,and to adopt a 16 s rRNA amplicon sequencing and metagenomic study strategy.Analytical tools to analyze the composition of bacterial vaginosis in women of childbearing age and the impact of dynamic observations on the efficacy of BV therapy,to predict the differences in functional genes of vaginal flora among BV patients and normal women,and to provide scientific basis for exploring the etiology and comprehensive prevention and treatment of BV.MethodsThis study was selected from the gynecological outpatients of the Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine for gynecological sampling and placed in a specimen collection tube and kept sealed at-20°C.Diagnosis of BV was performed using the Nugent score + Amsel method(gold standard).Then 17 samples of bacterial vaginosis,21 simple mildew infections,and 30 normal reproductive age women were randomly selected from the sample to extract the DNA of the vaginal secretion genome.The DNA was then submitted to the GENEWIZ company in Suzhou and sequenced through the Illumina platform to obtain the raw sequencing data.data.After obtaining the original data,remember to check the data for unqualified quality,check the quality of the original sequencing data by the md5 check batch command provided by the sequencing company,make the mapping file and check its format,and modify the barcode sequence,after which the subsequent processing in order to facilitate Qiime To splicing all fasta files,it is necessary to merge them into one file and also to modify the forward sequencing file after the barcode sequence.Next,the bar code sequence is extracted from the original reads information,and a barcodes.fastq file is generated and used together with the merged fastq file output.fastq and the metadata file map2.txt as the next cut library operation.The input file,followed by the cut library,is to classify each sequence into a different sample(specimen)according to the barcode file.The resulting file is a fasta formatted(*.fna)sequence file,all of which are classified.The sequence has a unique number and sample attribution information that is recoded,and an OTU table file is finally generated.After picrus was installed,the OUT table was normalized and the 16 S amplicon sub-macrogenomic function was predicted using picrust,and STAMP software was used to analyze differences between groups(samples).Results1.There were statistically significant differences in the abundance of 2233 genes in BV,MV and normal controls.Only the difference in abundance of 67 functional genes between high and low age groups was statistically significant.2.The average abundance of each gene between the BV and MV groups was compared with the average abundance of 248 genes.The difference was statistically significant.A representative three genes are highly expressed in the MV group and low in the BV group.Annotated as k02032: peptide/nickel transport system ATP-binding protein,k02033/4: peptide/nickel transport system permease protein.3.There was a statistically significant difference in the average abundance of 378 genes between the BV and Normal groups.In the BV group,high expression was observed.In the normal control group,the low expression gene codes were k00648,k00876,k03712,k06201,and k07088,respectively named as 3-acyl acyl carrier protein synthetase III,uridine cytidine kinase,and MarR family.Transcription regulator,copper balance protein and an unidentified protein;low expression in BV group,the high expression gene codes in the normal control group were k00077,k01478,respectively named 2-dehydroPantoate(2-dehydropantoate))and arginine deiminase(arginine deiminase).4.There was a statistically significant difference in the average abundance of 458 genes between MV and Normal groups.There are 10 representative genes which have differences between MV and normal control groups,and the gene codes are K07088,K01182,K00872,K00003,K00611,K00940,K00926,K01462,K00526,K01835.5.A gene with no annotation information in the KEEG library was found.The average abundance in the case group(BV and MV)was significantly higher than that in the control group,and its gene function deserved further study.Conclusions1.The three groups of BV,MV and Normal expressed differently in genomics.2.Gene k07088,without annotation information in the KEGG library,was highly expressed in the MV when comparing MV and Normal groups.When BV and Normal were compared,they were highly expressed in BV.3.Gene k07315 was expressed in BV,MV and normal controls.