Metagenomics Deep Sequencing In A Longevity Family In Hainan

ObjectiveBased on the method of deep sequencing of metagenomics,the structural characteristics of intestinal microbes in a long-lived family in Chengmai County,Hainan Province were studied.The structure and function of intestinal microflora related to longevity family were analyzed.From the view of intestinal microecology,it provides a possible theoretical basis for the basic research of longevity mechanism.MethodsThe feces of nine long-lived elderly in Chengmai County,Hainan Province were tested by high-throughput metagenomics deep sequencing technique,and the characteristics of intestinal microorganisms in long-lived families were analyzed.To explore potential biological markers associated with longevity by comparing the factors with the intestinal flora,such as genetic background,living environment,age.Results1.9 elderly people with an average age of 86.78±6.12 years old came from 6 families of the longevity family,including 5 males.They were healthy elderly through physical examination and medical history inquiry.The fecal samples were collected for metagenomics deep sequencing.A total of 475.6G sequencing data were obtained by using Illumina Hiseq2500 platform and the sequencing depth was 50-100G/samples.The species annotation for five dominant bacteria of the 9 elderly men showed that at the phylum level were Firmicutes(41.99%),Bacteroidetes(30.63%),Viruses noname(14.83%),Proteobacteria(6.32%);Actinobacteria(2.54%).While at the genus level the five dominant bacteria were Tunalikevirus(13.55%),Eubacterium(12.56%)Bacteroides(11.99%),Prevotella(9.91%)and Faecalibacterium(4.25%).2.Using Spearman Rank correlation analysis,13 species of intestinal microbes in the longevity family have correlation with age(P<0.05).Clostridium bartlettii,Clostridium celatum,Clostridium perfringens,Clostridium asparagiforme,Citrobacter freundii,Escherichia fergusonii,Bacillus cereus thuringiensis,and Acinetobacter baumannii were positively correlated with age.Five species were negatively correlated with age,they were Lacnospiraceae bacterium 1 1 57FAA,Ruminococcus albus,Enterobacteria phage HK225,Bacteroides oleiciplenus and Acidaminococcus fermentans.The species with the greatest weight was Clostridium bartlettii,with a correlation coefficient p=0.933(P=0.001).Based on the analysis of strain level in single nucleotide polymorphisms,three species were found to reflect age differences at the species level by PERMANOVA test,including Eubacterium rectale(R=0.72,P=0.008),Cray Klebsiella phage KP36(R=0.57,P=0.065),Bacteroides vulgatus(R=0.82,P=0.076).3.Grouping by different families and using principal co-ordinates analysis(PCoA),it was found that the composition of intestinal microbes in the same family/couple was similar(P=0.006).At the strain level based on single nucleotide polymorphisms analysis,7 species were found at the species level by PERMANOVA test to explain differences among different families,including Prevotella copri(R=0.85,P=0.006),Dorea longicatena(R=0.96,P=0.016),Bacteroides uniformis(R=0.85,P=0.006),Prevotella stercorea(R=0.97,P=0.016),Collinsella aerofaciens(R=0.78,P=0.016),Copulococcus catus(R=0.65,P=0.036),Methanobrevibacter smithii(R=0.95,P=0.043).4.Using DIAMOND method to compare the non-redundant reference gene sets to several public functional gene databases,the gene abundance information of the functional annotations of the elderly intestinal microbes was obtained.Using PERMANOVA test,the gene abundance in the virulence factor database 1,carbohydrate active enzyme database 2 can explain the differences between different families(R1=0.72 P1=0.047,R2=0.73 P2=0.016).5.175 metabolites were found by using NJS16 collaborative network construction method to analyze the potential of intestinal microbes to metabolites in longevity family.Using Spearman rank correlation analysis,it was found that the potential of 11 intestinal microbes to metabolites were related to age(P<0.05).The affected metabolites included L-Asparagine,histamine,4-Aminobenzoate,alpha-Linolenic acid,Acetone,Glycochenodeoxycholate,Glycocholate,Glycine Glycodeoxycholate,Glycolithocholate,Butyrate and cis-Aconitate.The metabolite with the greatest potential was L-Asparagine,with correlation coefficient p=0.801,P=0.0095.ConclusionThe deep-sequence metagenomics technique was used to analyze the longevity family in Hainan,showing the characteristics of the intestinal flora of the elderly in the longevity family in Hainan,and 16 species related to longevity were found.The difference analysis showed that the similarity between the spouses was higher.It was found that phage is common in long-lived old people and may be another potential direction for studying the longevity/aging mechanism.