Extensive profiling of a complex microbial community by high-throughput sequencing - PubMed (original) (raw)

Extensive profiling of a complex microbial community by high-throughput sequencing

Janet E Hill et al. Appl Environ Microbiol. 2002 Jun.

Abstract

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.

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Figures

FIG. 1.

FIG. 1.

(A) Frequency distribution of unique nucleotide sequences recovered from the combined pig feces cpn60 libraries. (B) Taxonomic breakdown of total library contents. Assignment to a taxonomic group was based on comparisons of clone sequences to a database of cpn60 reference sequences.

FIG. 2.

FIG. 2.

Phylogenetic relationships of 280 unique Cpn60 peptide sequences translated from 398 unique nucleotide sequences. Distance calculations were made by the Dayhoff PAM matrix, and the dendrogram was produced by neighbor joining. The scale bar represents 0.1 substitution per site. Branches are colored according to the assigned taxonomic group of the sequences (red, CFB group; green, Proteobacteria gamma; blue, Bacillus/Clostridium group; orange, Proteobacteria beta; pink, spirochetes; black, other).

FIG. 3.

FIG. 3.

Phylogenetic relationships of 12 clone peptide sequences assigned to the CFB group, including the two most abundant cloned sequences (represented by 001_f12 and 002_a03). The tree is a consensus of 100 neighbor-joined trees. Distance calculations were made by the Dayhoff PAM matrix, and branch lengths were imposed on the consensus tree by Fitch. Nodes with bootstrap values of >50% are indicated with white dots. Reference sequences used in the tree are Flavobacterium hydatis (GenBank accession no. AAK32145), Flavobacterium ferrugineum (AAK32146), Bergeyella zoohelcum (ATCC 43767), Chryseobacterium meningosepticum (ATCC 13253), Chryseobacterium gleum (ATCC 35910), Bacteroides forsythus (CAB43992), Bacteroides vulgatus (ATCC 8482), Bacteroides uniformis (ATCC 8492), Bacteroides ovatus (ATCC 8483), Prevotella bivia (ATCC2 9303), Prevotella intermedia (ATCC 25611), Rhodothermus marinus (strain ITI 376, AAD37976), and Chlorobium tepidum (derived from contig 3499, TIGR unfinished genome database).

FIG. 4.

FIG. 4.

Cumulative frequency distribution plots for the pig feces library (solid line), a population of individual species from 172 different eubacterial and eukaryotic genera, a single taxonomic subclass (77 species from 34 genera of Proteobacteria gamma), and a single genus, Lactobacillus. Plots were generated from DNA identity matrices derived from ClustalW multiple sequence alignments by GeneDoc.

FIG. 5.

FIG. 5.

(A) Frequency distributions of unique nucleotide sequences recovered from clone libraries P56, P40, B40, and B56. (B) Taxonomic composition of libraries B40, B56, P40, and P56.

FIG. 6.

FIG. 6.

Taxonomic composition of groups of library clones pooled by PCR annealing temperature used in library construction (left panel) and genomic DNA template extraction method (right panel).

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