Research On The Relationship Between High-resolution Human Intestinal Metagenome And Diseases

In recent years,gut dysbiosis has been associated with numerous diseases,including metabolic diseases,digestive tract diseases and immune diseases.Most of the existing metagenomic studies focus on the structure and abundance of microbiota at species level(or above the level,such as family and genus)to study the relationship between the gut microbes and human health.However,different strains within a species may exhibit high variability in terms of function,despite that they are similar in genomic contents.Researchers have gradually realized that it is necessary to go deep into the strain level to study on the relationship between the gut metagenome and diseases.The strain diversity mainly originates from the genomic variation of microorganisms,including single nucleotide polymorphisms(SNP),short insertions/deletions(Indel)and structural variation(SV).SNPs are the most abundant type of metagenomic variation,and the number of Indel is about two orders of magnitude smaller than SNP,while the number of SV is even smaller.The study of metagenomic variation,especially metagenomic SNPs,is a central approach to understanding microbial community-host interactions at the strain level.Although there are few studies on the relationship between metagenomic variation and diseases in the world,some important discoveries have been published.For example,in the field of type 2 diabetes(T2D),specific strain of Bacteroides coprocola has been found to be associated with the development of T2D,and the intestinal specific strains promoted by dietary fiber can effectively alleviate T2D.Identifying strains that are clearly associated with disease occurrence and progression is crucial for precision microbiota therapy.As an emerging research direction,strain level analysis still needs to be explored: How much sequencing size is sufficient for strain-level analysis? How to disentangle accurate strains from metagenomic sequencing data? When the sequencing depth is sufficient,is it possible for us to further discover the relationship between strain-specific genes and diseases?To answer these questions,we conducted the study "Research on the relationship between high-resolution human intestinal metagenome and diseases".High resolution refers to the ability to achieve resolution accuracy at the strain level,which is different from the current research accuracy that only reaches the genus,and species level.This research contains three parts,the first is to answer the sufficient sequencing depth required for systematic SNP analysis of human intestinal metagenome,the second is to analyze the relationship between intestinal metagenome and liver cirrhosis at the strain level,and the third is to study the relationship between SNPs of intestinal metagenome glycoside genes and T2D under ultra-deep sequencing depth.In the first part,the sufficient sequencing depth required for systematic SNP analysis of human intestinal metagenome.Metagenomic SNPs are important indicators of strain-level complexity and have been used to disentangle different strains from the metagenome.Currently,sequencing size of around 6-9 GB(Gigabases)is commonly used in mainstream metagenomic studies,whether it is enough to study at the strain level remains unkown.We conducted ultra-deep sequencing of the human gut microbiome and constructed an unbiased in-house framework to perform a reliable SNP analysis.SNP landscape and SNP function profiles of the human gut metagenome by ultra-deep sequencing were obtained.In addition,the relative abundance,sequencing coverage,sequencing depth,the number of SNPs and the coding function of the SNP-located genes of dominant strains from conventional and ultra-deep sequencing were thoroughly compared,and the relationship between SNPs and sequencing depth were investigated.The results show that ultra-deep sequencing could detect functionally important SNPs and new strain genomes.When the sequencing size was small,the evolutionary indicators inferred from the data could not reflect true selection pressure in the environment.This suggests that the conventional sequencing size may not be sufficient to support systematic metagenomic SNP investigations.We also constructed a machine learning model SNPsnp(https://github.com/labomics/SNPsnp)to provide guidance for researchers to determine the optimal and affordable sequencing depth for their projects.To conclude,this study suggests the importance of evaluating sequencing depth before starting SNP analysis and provides new ideas and references for future strain-level investigations.In the second part,strain-level analysis of the gut metagenome and its relationship with liver cirrhosis.Nowadays,the strain diversity of species and its association with liver cirrhosis(LC)have received little attention.We constructed a computational framework for intestinal metagenomic strain level analysis and explored the strain diversity of the gut microbes in patients with LC and healthy individuals.We found that only Faecalibacterium prausnitzii shows different SNP patterns between the LC and HC groups.Strain diversity analysis showed that although most genomes of F.prausnitzii are more deficient in the LC group than in the HC group at the strain level,which is the same as the result at the species level,a subgroup of 19 F.prausnitzii strains showed no sensitivity to LC.The functional differences between this subgroup and other strains may involve short-chain fatty acid production and chlorine-related pathways.These findings demonstrate functional difference among F.prausnitzii subgroups.The results also suggest that not all F.prausnitzii strains are directly associated with liver cirrhosis,and only some strains may serve as potential probiotics or intervention targets for treatment for liver cirrhosis,which is different from previous knowledge.Our results extend current knowledge about strain heterogeneity and relationships between F.prausnitzii and LC at high-resolution strain level,which suggests the need of strain level analysis to uncover the relationship between intestinal metagenomics and diseases.In the third part,the analysis of metagenomic SNPs of glycosidase genes and its relationship with T2D.We have found that a single SNP of a glycosidase gene in B.coprocola is associated with T2D in previous studies.To explore the relationship between SNPs of glycosidase family genes and T2D,we obtained 487 glycosidase genes of the gut microbiota based on the NCBI(National Center for Biotechnology Information)database and the high-quality human intestinal microbiome genome databases.Targeting these gene regions,we designed targeted capture probes to efficiently capture intestinal microorganism glycosidase genes,sequenced 94 samples,and established the corresponding sequencing evaluation method.At the same time,to eliminate the interference caused by common human intestinal microorganisms,we constructed a non-redundant reference genes set,and obtained the SNP profiles of glycosidase genes in the T2D patients and the healthy control(HC)group.Through comparison analysis from dimension of single glycosidase gene,amino acid change,glycosidase family and bacteria taxonomy level between the T2D patients and HC group,we analyzed the SNPs of multi-dimensional intestinal microbe glycosidase genes and its relationship with T2D.To conclude,we explored solutions to key problems in the field of high-resolution metagenomic research from three aspects.We built a machine learning model named SNPsnp by modeling the SNP profiles of ultra-deep metagenomic sequencing and provide guidance for researchers to determine the required sequencing depth for their project.The establishment of intestinal metagenomic strain-level analysis framework expands the understanding of the relationship between strain diversity of F.prausnitzii and LC.Targeted capture sequencing was used to study the diversity of specific bacteria genes,and the relationship between SNPs of intestinal microbe glycosidase genes and T2D was analyzed from a multidimensional perspective.In this study,we explored intestinal metagenomic SNPs from multiple dimensions,which expands the current knowledge of intestinal microbial genomic variation and strain diversity,and provides new ideas for high-resolution metagenomic research.What’s more,this study also provides valuable perspectives for future studies on metagenomic strain diversity of other organs or other environments.