File S1 - Polyphasic Analysis of a Middle Ages Coprolite Microbiota, Belgium

Supporting information. Note S1. Specific PCR-amplifications and Sanger-sequencing, quantitative real time PCR. Table S1. Typical gastro-intestinal, environmental and pathogenic bacteria assigned to the coprolite metagenome#. #Metagenomic reads were blasted against the NCBI protein database using a translated nucleotide query. Underrepresented taxonomic genera are not shown. Table S2. Number and size of contigs that were assigned to (A) Bacteroides spp. and (B) to bacterial pathogens associated to the coprolite sample#. #The contig identifier, its length (bp) and the annotation according to the best BLAST hit (BLASTX versus the non-redundant NCBI database, E-value<1e−05) are summarized. The E-value, the hit accession identifier and the percent of identity are also provided. Contigs of °human and *pig gut microbiota Bacteroides species. Table S3. Cultured microorganisms#. # Reported are the culture conditions, the cultured microorganisms and the mode leading to species identification. Reported is also if the cultured bacteria were found in the high-throughput pyrosequencing dataset. ° When the identification was based on BLAST annotation of the amplified 16 S rRNA gene region additional phylogenetic trees were constructed (Figures S2). Table S4. Bacterial pathogens identified from the amplified 16 S rRNA V6 region#. # The species level was defined with a minimum sequence identity of 98.7% using BLAST similarity searches against RDP databases. Table S5. Primers used to amplify DNA from intestinal parasites, bacterial pathogens and amoebae. Table S6. The quantitative real-time PCR systems that were tested. Figure S1. Working overview of the polyphasic approach used to analyze the Namur coprolite. Figure S2. Phylogenetic of 16 S rRNA gene sequences generated form cultivated bacteria species. The tree was constructed using the PhyML algorithm with a bootstrap of 100. The bootstrap support is reported for each branch. Phylogenetic tree of 16 S rDNA amplicons closely related to (A) Bacillus horti, (B) Paenibacillus spp., (C) Rhodanobacter spp. and (D) Clostridium magnum. Figure S3. Phylogenetic tree of a hydrolase. A phylogenetic tree was generated from the translated open reading frame of a contig encoding a hydrolase close to Bordetella species. The tree was constructed using the PhyML algorithm with a bootstrap of 100. The bootstrap support is reported for each branch. Figure S4. Bayesian source-tracking results. (A) The mixture of taxa associated to the 16 S rDNA gene amplicon dataset of the coprolite specimen was compared to known dataset of various environments. To control the workflow used to perform the analyses, two known samples (B) one coprolite previously investigated [9] and (C) a soil sample [10] were positively tested. Figure S5. Phylogenetic tree of 16 S rDNA amplicons matching to Bartonella sp. The tree was constructed using the PhyML algorithm with a bootstrap of 100. The bootstraps are reported for each branch. Phylogenetic tree of 16 S rDNA amplicons closely related to (A) B. henselae, B. koehlerae and (B) B. quintana. Figure S6. Alignment and of the amplicon matching to Bordetella and Achromobacter. The sequence alignment was performed using CLUSTALW multiple alignment tool [11]. Figure S7. Metabolic comparison of modern metagenomes to the coprolite metagenome. The Principal coordinates analysis was based on read classification according to BLASTX searches against the SEED Database. For each metagenome included the MG-RAST accession number is given. Compared metagenomes are from soil (yellow cluster), healthy mammalian and human feces (blue cluster); and the coprolite (red). The coprolite metagenome does not group with either the modern gut or soil microbiota.
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