Study On The New Method For High-throughput Bacterial Classification And Its Application In The Discovery Of Marine Novel Bacteria

Prokaryotes are abundant and widely distributed in nature and participate in the biogeochemical cycles of various elements.At present,the vast majority of prokaryotes cannot be cultured in the laboratory,and the discovery of novel microbial species and groups is at a low speed.It is of great significance to accelerate the discovery and classification of novel microbial species for uncovering the microbial diversity.The classification and identification of novel microbial species are performed through polyphasic taxonomy,and genome-based classification methods are evolving rapidly.The above classification methods are labor-intensive and expensive in large-scale classification,which have become the bottleneck for the discovery of novel species.To address the above problem,this study developed a new method for high-throughput,low-cost bacterial classification and identification,and applied this new method to the large-scale culture study of marine bacteria.The details are as follows.Ⅰ.A new high-throughput and low-cost bacterial classification and identification method was designed.The main idea of this method is to classify and identify bacteria based on their genome sequences,so as to achieve high-precision classification,and to reduce the overall cost of genome acquisition and simplify experimental procedures by mixing the culture before sequencing.The main steps are as follows:(1)picking and mixing bacterial colonies,(2)library preparation and high-throughput sequencing,(3)assembling and binning the genomes,and(4)classification based on genome sequences.Ⅱ.Bacteria from coastal sediment were cultured at a large scale.A surface sediment sample was collected from the coastal area of Qingdao and 13 kinds of solid media were used for the large-scale cultivation of bacteria.A total of 105,724 colonies were obtained.A minor portion of colonies were randomly picked for 16S rRNA gene sequencing,and 221 sequences were obtained.Cluster analysis and EzBioCloud analysis showed that these sequences belonged to 5 phyla,8 classes,18 orders,31 families,85 genera and 126 species,including 32 potential novel species.Ⅲ.The new method was applied to classify and identify coastal sediment bacteria at a large scale.From the above large-scale culture,79,140 colonies were selected for classification using the new method.A total of 1,816 Gb data were obtained by highthroughput sequencing,and a total of 1,681 dereplicated genomes(completeness≥50%.contamination≤10%)were obtained after assembling and binning.Clustering of these genomes yielded 1,110 species(ANI=0.95,AF=0.30).Classification with GTDB-Tk software revealed up to 924 potential new species.This number is equivalent to 4%of all currently recognized prokaryotic species and 2%of the species in the lastest version of Genome Taxonomy Database(GTDB),which includes genomes from pure culture,metagenome-assembled genomes(MAGs)and single-cell genomes.In addition,phylogenetic analysis based on 120 universal single-copy bacterial marker genes revealed two new orders from the above genomes,one belonging to the class Alphaproteobacteria and the other belonging to the class Gammaproteobacteria.In conclusion,a new high-throughput and low-cost method for bacterial classification and identification was developed in this study.The method was applied to a large-scale culture study of bacteria from the coastal surface sediment,and 924 potential novel species were uncovered from one single sediment sample.The wide application of the new method is expected to greatly accelerate the pace of the discovery of novel microbial species and promote the studies of microbial diversity and resource discovery.