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Research Article Review Jmb J. Microbiol. Biotechnol. (2017), 27(0), 1–7 https://doi.org/10.4014/jmb.1707.07027 Research Article Review jmb Methods 20,546 sequences and all the archaeal datasets were normalized to 21,154 sequences by the “sub.sample” Bioinformatics Analysis command. The filtered sequences were classified against The raw read1 and read2 datasets was demultiplexed by the SILVA 16S reference database (Release 119) using a trimming the barcode sequences with no more than 1 naïve Bayesian classifier built in Mothur with an 80% mismatch. Then the sequences with the same ID were confidence score [5]. Sequences passing through all the picked from the remaining read1 and read2 datasets by a filtration were also clustered into OTUs at 6% dissimilarity self-written python script. Bases with average quality score level. Then a “classify.otu” function was utilized to assign lower than 25 over a 25 bases sliding window were the phylogenetic information to each OTU. excluded and sequences which contained any ambiguous base or had a final length shorter than 200 bases were Reference abandoned using Sickle [1]. The paired reads were assembled into contigs and any contigs with an ambiguous 1. Joshi NA, FJ. 2011. Sickle: A sliding-window, adaptive, base, more than 8 homopolymeric bases and fewer than 10 quality-based trimming tool for FastQ files (Version 1.33) bp overlaps were culled. After that, the contigs were [Software]. further trimmed to get rid of the contigs that have more 2. Schloss PD. 2010. The Effects of Alignment Quality, than 1 forward primer mismatch and 2 reverse primer Distance Calculation Method, Sequence Filtering, and Region on the Analysis of 16S rRNA Gene-Based Studies. mismatch. The primer sequences were trimmed off. Then Plos Comput. Biol. 6: e1000844. the sequences that were the same with each other were 3. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss merged as one unique sequence to accelerate the filtering PD. 2013. Development of a Dual-Index Sequencing Strategy calculation. The remaining unique sequences were aligned and Curation Pipeline for Analyzing Amplicon Sequence using SILVA bacterial and archaeal reference database for Data on the MiSeq Illumina Sequencing Platform. Appl. bacterial and archaeal datasets separately. Afterwards, the Environ. Microb. 79: 5112-5120. sequences that did not align to the correct region were 4. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. 2011. excluded [2]. Both ends of the sequences were trimmed to UCHIME improves sensitivity and speed of chimera ensure that all the sequences started and ended at the same detection. Bioinformatics 27: 2194-2200. position. Additionally, a preclustering algorithm was 5. Mizrahi-Man O, Davenport ER, Gilad Y. 2013. Taxonomic employed to merge sequences by allowing 1 bp difference Classification of Bacterial 16S rRNA Genes Using Short for every 100 bp [3]. Chimeras were screened and removed Sequencing Reads: Evaluation of Effective Study Designs. Plos One. 8: e53608. from the resulting sequences using Uchime packaged in Mothur [4]. All the bacterial datasets were normalized to A 2017 ⎪ Vol. 27⎪ No. 0 2Name et al. Table S1. The taxonomic information and the proportion of all the 98 OTUs in the core bacterial community. Abundant Abundant Groups OTU ID Percentage in N FS in N SS phylum class order family genus samples samples Group 1 Otu00001 4.31% 21 15 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Otu00002 4.20% 17 14 Firmicutes Negativicutes Selenomonadales unclassified_ unclassified_ Selenomonadales Selenomonadales Otu00003 3.51% 18 17 Thermotogae Thermotogae Thermotogales Thermotogaceae Mesotoga Otu00004 3.24% 21 17 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Proteiniphilum Otu00005 2.67% 21 17 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Macellibacteroides Otu00006 2.67% 21 17 Synergistetes Synergistia Synergistales Synergistaceae Aminivibrio Otu00007 2.02% 21 17 Bacteroidetes Bacteroidia Bacteroidales Rikenellaceae vadinBC27_wastewater- sludge_group Otu00017 0.92% 17 17 Synergistetes Synergistia Synergistales Synergistaceae Aminivibrio Otu00021 0.76% 21 14 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides Otu00023 0.75% 17 16 Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae Desulfovibrio Otu00028 0.59% 18 17 Spirochaetae Spirochaetes unclassified_ unclassified_ unclassified_ Spirochaetes Spirochaetes Spirochaetes Otu00052 0.34% 18 14 Synergistetes Synergistia Synergistales Synergistaceae unclassified_ Synergistaceae Group 2 Otu00011 1.42% 21 12 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Otu00013 1.28% 21 11 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides Otu00016 0.98% 21 9 Spirochaetae Spirochaetes Spirochaetales PL-11B10 unclassified_PL-11B10 Otu00025 0.75% 21 8 Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae Desulfovibrio Otu00030 0.66% 20 2 Firmicutes Negativicutes Selenomonadales Veillonellaceae Veillonella Otu00033 0.61% 21 9 Firmicutes Clostridia Clostridiales Lachnospiraceae Clostridium XlVa Otu00036 0.54% 21 3 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Otu00038 0.50% 18 4 Spirochaetae Spirochaetes Spirochaetales Spirochaetaceae Spirochaeta Otu00041 0.43% 17 3 Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae Desulfovibrio Otu00044 0.44% 18 2 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Otu00048 0.33% 17 3 Bacteroidetes Bacteroidia Bacteroidales Rikenellaceae Blvii28_wastewater- sludge_group Otu00049 0.38% 21 1 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Otu00064 0.29% 19 1 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Group 3 Otu00008 2.26% 5 17 Chloroflexi Anaerolineae Anaerolineales Anaerolineaceae unclassified_ Anaerolineaceae Otu00009 2.07% 8 17 Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobacteraceae unclassified_ Desulfobacteraceae Otu00010 1.95% 13 17 Chloroflexi Anaerolineae Anaerolineales Anaerolineaceae unclassified_ Anaerolineaceae Otu00014 1.19% 9 17 Chloroflexi Anaerolineae Anaerolineales Anaerolineaceae unclassified_ Anaerolineaceae Otu00015 1.05% 16 16 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus Otu00018 0.82% 9 17 Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophaceae Smithella(84) Otu00019 0.89% 5 17 Synergistetes Synergistia Synergistales Synergistaceae Thermovirga Otu00020 0.82% 12 16 Bacteroidetes Sphingobacteriia Sphingobacteriales WCHB1-69 unclassified_ WCHB1-69 Otu00022 0.80% 3 17 Proteobacteria Betaproteobacteria Rhodocyclales Rhodocyclaceae unclassified_ Rhodocyclaceae Otu00024 0.80% 2 16 Firmicutes Clostridia Clostridiales Syntrophomonadaceae Syntrophomonas Otu00026 0.78% 2 17 Bacteroidetes vadinHA17 unclassified_ unclassified_ unclassified_ vadinHA17 vadinHA17 vadinHA17 Otu00029 0.72% 3 17 unclassi- unclassified_Bacteria unclassified_Bacteria unclassified_Bacteria unclassified_Bacteria fied_Bacteria Otu00031 0.65% 16 16 Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Brachymonas Otu00034 0.61% 14 17 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Proteiniphilum Otu00035 0.59% 1 16 unclassified_ unclassified_ unclassified_Bacteria unclassified_Bacteria unclassified_Bacteria Bacteria Bacteria J. Microbiol. Biotechnol. Title 3 Table S1. The taxonomic information and the proportion of all the 98 OTUs in the core bacterial community. Otu00039 0.54% 2 17 Bacteroidetes vadinHA17 unclassified_ unclassified_ unclassified_ vadinHA17 vadinHA17 vadinHA17 Otu00040 0.42% 14 17 Spirochaetae Spirochaetes LNR_A2-18 unclassified_LNR_A2-18 unclassified_ LNR_A2-18 Otu00042 0.48% 1 17 Proteobacteria Deltaproteobacteria unclassified_ unclassified_ unclassified_ Deltaproteobacteria Deltaproteobacteria Deltaproteobacteria Otu00043 0.46% 8 17 Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Limnobacter Otu00046 0.40% 9 17 Spirochaetae Spirochaetes LNR_A2-18 unclassified_LNR_A2-18 unclassified_LNR_A2-18 Otu00051 0.39% 4 16 Synergistetes Synergistia Synergistales Synergistaceae Aminobacterium Otu00055 0.35% 13 16 Synergistetes Synergistia Synergistales Synergistaceae Aminobacterium Otu00057 0.30% 13 16 Spirochaetae Spirochaetes LNR_A2-18 unclassified_LNR_A2-18 unclassified_LNR_A2-18 Otu00059 0.33% 6 16 Spirochaetae Spirochaetes LNR_A2-18 unclassified_LNR_A2-18 unclassified_LNR_A2-18 Otu00061 0.31% 2 17 Proteobacteria unclassified_ unclassified_ unclassified_ unclassified_ Proteobacteria Proteobacteria Proteobacteria Proteobacteria Otu00062 0.31% 1 15 Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophaceae Smithella Otu00067 0.27% 1 17 Proteobacteria Betaproteobacteria Hydrogenophilales Hydrogenophilaceae Thiobacillus Otu00068 0.25% 8 15 Firmicutes Clostridia Clostridiales Lachnospiraceae Anaerostipes Otu00069 0.27% 0 15 Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae Hyphomicrobium Otu00071 0.26% 7 15 Proteobacteria Betaproteobacteria Neisseriales Neisseriaceae unclassified_ Neisseriaceae Otu00075 0.26% 0 16 Firmicutes Clostridia Clostridiales Peptostreptococcaceae Incertae_Sedis Otu00076 0.25% 4 15 Firmicutes Bacilli Bacillales Paenibacillaceae Brevibacillus Otu00078 0.25% 0 15 Proteobacteria Gammaproteobacteria Run-SP154 unclassified_Run-SP154 unclassified_ Run-SP154 Otu00079 0.23% 0 15 Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae Hyphomicrobium Otu00080 0.23% 4 16 Candidate_ unclassified_ unclassified_
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