Summary: the Supplementary Materials Include Four Figures and Six Tables That Are Listed Below

Summary: The supplementary materials include four figures and six tables that are listed below.

Figures Figure S1: (a-c) Rarefaction curves of all samples (a), healthy control samples (b) and samples of CRC patients (c); (d-f) Shannon diversity index curves of all samples (d), healthy control samples (e) and samples of CRC patients (f). Blue lines represent healthy control samples; red lines represent samples of CRC patients.

Figure S2: Relative contributions of dominant phyla in the gut microbiota of healthy volunteers (a) and CRC patients (b).

Figure S3: The numbers of OTUs identified at several different similarity levels. The x-axis shows the identity, the y-axis shows the number of OTUs.

Figure S4: PCA scores plots based on the relative abundance of OTUs at different similarity levels. (a): 95% similarity level; (b): 97% similarity level; (c): 99% similarity level; (d): 100% similarity level; Blue circles represent healthy volunteers; red circles represent CRC patients. Tables Table S1. Primers and amplification conditions for qPCR assays in this study Target Primers Primer Sequences References Amplification conditions bacterial group Total bacteria Uni-1 (331F) 5’-TCCTACGGGAGGCAGCAGT-3’ (Nadkarni et al., Initial denaturing at 95°C for 4 min; 40 cycles of denaturing 2002) at 95°C for 15 s, and annealing/extension at 60°C for 1 min. Uni-2 (797R) 5’- GGACTACCAGGGTATCTAATCCTGT T-3’ Bacteroides Bfr-F 5’-CTGAACCAGCCAAGTAGCG-3’ (Li et al., 2009) Initial denaturing at 95°C for 2 min; 40 cycles of denaturing spp. at 95°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 1 min. Bfr-R 5’- CCGCAAACTTTCACAACTGACTTA- 3’ Butyrate BCoATscrF 5’- (Louis & Flint, Initial denaturing at 95°C for 3 min; 40 cycles of denaturing producing GCIGAICATTTCACITGGAAYWSITG 2007) at 95°C for 30 s, annealing at 53°C for 30 s, and extension bacteria GCAYATG-3’ at 72°C for 30 s. BCoATscrR 5’- CCTGCCTTTGCAATRTCIACRAANG C-3’ Table S2. Numbers of total sequences, unique sequences after CD-HIT clustering/NAST alignment, and number of OTUs (98% similarity level) in each sample Healthy volunteers CRC patients Subject Total Unique OTUs Subject Total Unique OTUs name sequences sequences (98%) name sequences sequences (98%) H01 3291 756 269 C01 3293 687 224 H02 3031 698 272 C03 2790 530 160 H03 3255 671 257 C05 3534 706 225 H04 2961 572 195 C07 3309 702 242 H05 3414 747 233 C09 2670 561 192 H06 2307 396 150 C11 2945 600 213 H07 2361 607 245 C13 2874 756 293 H08 2318 358 130 C15 2795 681 252 H09 1462 433 171 C17 2748 668 221 H10 2461 545 195 C19 2164 568 202 H11 2564 641 237 C21 2459 395 122 H12 2323 497 173 C23 2971 611 186 H13 2556 658 237 C25 3997 940 338 H14 1774 443 186 C27 2848 663 235 H15 2180 445 128 C29 2592 440 171 H16 2448 724 269 C31 2699 611 215 H18 2523 517 168 C33 3582 744 251 H19 2058 486 176 C35 2367 419 118 H20 2792 569 211 C37 1332 394 146 H21 2734 667 253 C39 3570 428 147 H22 2665 568 216 C41 3138 719 251 H23 2382 537 208 C43 3219 783 301 H24 2863 667 243 C45 2806 560 194 H25 2465 616 219 C47 2540 573 206 H26 2770 612 199 C49 2775 666 224 H27 2716 757 267 C51 2062 363 121 H30 2811 634 211 C53 2858 713 293 H32 1520 437 159 C55 2392 548 197 H33 2495 599 221 C57 3057 769 289 H34 2189 592 214 C59 3344 681 217 H35 2587 546 192 C61 3144 735 275 H36 2810 538 185 C63 2183 393 118 H37 2809 695 268 C65 2658 679 248 H38 3005 786 304 C67 2375 627 239 H39 2286 455 158 C69 2448 636 211 H40 2774 553 193 C71 2257 516 192 H41 2648 610 244 C73 2867 544 178 H42 3154 650 227 C75 3232 566 163 H43 2928 696 259 C77 3085 570 178 H44 3318 731 279 C79 3156 813 310 H45 2855 774 292 C81 3216 723 250 H46 2605 609 219 C83 3073 649 291 H47 2568 612 238 C85 3928 676 257 H48 2481 572 213 C87 2709 610 216 H49 2882 567 186 C89 3130 730 262 H50 2706 591 190 C93 2742 749 288 H50 2545 538 190 - - - - H52 2763 442 127 - - - - H53 2956 724 270 - - - - H54 3219 629 204 - - - - H55 2732 696 267 - - - - H56 2878 639 216 - - - - H57 2453 598 229 - - - - H58 2585 691 265 - - - - H59 2716 705 256 - - - - H60 1992 482 181 - - - -

Table S3. Comparison of diversity indices between the gut microbiota of healthy volunteers (H) and CRC patients (C); values are shown as mean ± standard deviation. H C p value

Shannon diversity index (H’) 3.62±0.45 3.56±0.47 0.510

Simpson’s diversity index (1-D) 0.93±0.04 0.93±0.04 0.885

Buzas and Gibson's evenness index (exp(H’) /S) 0.18±0.05 0.17±0.05 0.277

Table S4. Relative contribution, median and range of the predominant phyla in the gut microbiota of healthy volunteers (H) and CRC patients (C) Phylum Relative contribution (%) Median, range (%) H C H C Firmicutes 57.2 63.1 59.3, 29.6-87.2 63.9, 16.6-94.5 Bacteroidetes 32.0 22.7 31.0, 0.13-62.9 18.6, 0.17-75.0 Proteobacteria 2.81 4.68 1.81, 0.24-38.1 2.81, 0.29-28.5 Actinobacteria 2.22 4.55 1.10, 0-11.5 2.61, 0.04-31.1 Fusobacteria 2.20 1.59 0.04, 0-36.2 0.12, 0-34.5 Table S5. Phylogeny of the 48 key OTUs (98% similarity level) and p value of Mann-Whitney test between healthy volunteers (H) and CRC patients (C) OTU Taxonomical assignments (RDP Classifier)# Nearest neighboring Relative p value number bacterium/bacteria contribution (%) (Mann-Whitney Phylum Class Order Family Genus (similarity)* H C test) OTU0005 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Bacteroides vulgatus; 7.3955 2.0929 1.93E-04 AB510712 (100.0% ) OTU0945 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Bacteroides vulgatus; 0.0327 0.0083 5.87E-04 AB510712 (98.7%) OTU4160 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Bacteroides uniformis; 0.0041 0 0.023 AB510711 (97.5%) OTU3045 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Bacteroides fragilis (T); 0 0.0114 0.006 ATCC 25285; CR626927 (98.1%) OTU0185 Bacteroidetes Bacteroidia Bacteroidales Rikenellaceae Alistipes Alistipes indistinctus; 0.0504 0.0053 3.82E-04 AB490804 (97.5% ) OTU0845 Actinobacteri Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium Bifidobacterium 0.0041 0.0303 3.40E-04 a catenulatum (T); ATCC27539; AF432082 Bifidobacterium pseudocatenulatum; AY166521 Bifidobacterium thermacidophilum subsp. thermacidophilum; AY166524 Bifidobacterium angulatum; AB489093 (97.9%) OTU1336 Actinobacteri Actinobacteria Bifidobacterium 0.0007 0.0076 0.013 a pseudocatenulatum; AB125917 Bifidobacterium catenulatum (T); ATCC27539; AF432082 Bifidobacterium angulatum; AB489093 (95.1%) OTU0176 Firmicutes Clostridia Clostridiales Ruminococcaceae Oscillibacter Oscillospira 0.0443 0.0068 7.14E-04 guilliermondii; AB040499 (97.1% ) OTU0039 Proteobacteri Gammaproteobacteri Enterobacteriales Enterobacteriaceae Escherichia/Shigella Shigella dysenteriae 0.9398 2.8150 2.42E-05 a a (T); X96966 Escherichia coli; X80731 Hafnia alvei; Z83203 Shigella sonnei; X80726 (100.0%) OTU0484 Proteobacteri Gammaproteobacteri Enterobacteriales Enterobacteriaceae Escherichia/Shigella Hafnia alvei; Z83203 0 0.0061 0.006 a a Escherichia albertii (T); LMG 20976; AJ508775 Shigella boydii;AY696670 Escherichia coli; CP000243 (99.4%) OTU1955 Proteobacteri Gammaproteobacteri Enterobacteriales Enterobacteriaceae Escherichia/Shigella Escherichia 0.0007 0.0083 0.006 a a coli;X80731 Hafnia alvei; Z83203 (98.8%) OTU2525 Proteobacteri Gammaproteobacteri Enterobacteriales Enterobacteriaceae Klebsiella oxytoca; 0.0014 0.0121 0.010 a a FM207542 (97.0%) OTU1203 Proteobacteri Gammaproteobacteri Escherichia fergusonii; 0.0041 0.0174 0.012 a a AJ277981 (97.000% similar) OTU0901 Proteobacteri Gammaproteobacteri Pseudomonadale Moraxellaceae Acinetobacter Acinetobacter 0.0007 0.0091 0.012 a a s johnsonii; DQ257432 (100.0%) OTU3671 Proteobacteri Gammaproteobacteri Enterobacteriales Enterobacteriaceae Citrobacter freundii; 0 0.0046 0.012 a a FJ542329 (98.8%) OTU1380 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium Fusobacterium 0.0020 0.0614 0.005 periodonticum; AJ810271; Fusobacterium nucleatum E9_12; AF481217 (100.0%) OTU1801 Actinobacteri Actinobacteria Coriobacteriales Coriobacteriaceae Eggerthella Eggerthella lenta (T); 0.0014 0.0121 0.009 a ATCC25559; AF292375 (97.9%) OTU0147 Firmicutes Clostridium spiroforme; 0.8424 0.4377 0.009 X73441 (94.4%) OTU0391 Firmicutes Bacilli Bacillales Staphylococcaceae Gemella Gemella morbillorum; 0.0068 0.0728 0.006 GU561406 (100.0%) OTU1407 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus Enterococcus ratti; 0 0.0190 0.003 AF326472 Enterococcus faecium; AB018210 Enterococcus faecalis; EU717961 Enterococcus durans; EU717964 (98.8%) OTU2673 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus Enterococcus faecium 0 0.0038 0.012 (T); LMG 11423; AJ301830 Enterococcus durans (T); DSM20633; AJ276354 Enterococcus pseudoavium; AJ301837 Enterococcus hirae; AJ276356 (99.4%) OTU1174 Firmicutes Clostridia Clostridiales Clostridium innocuum 0.0007 0.0228 0.005 (T); M23732 (99.4%) OTU1435 Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus Streptococcus 0.0007 0.0106 0.011 thermophilus (T); ATCC 19258; AY188354 (99.3%) OTU1803 Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus Streptococcus 0 0.0053 0.012 gallolyticus (T); LAB617; Z94012 (97.6%) OTU0432 Firmicutes Clostridia Clostridiales Veillonellaceae Phascolarctobacteriu Phascolarctobacterium 0.0259 0.0091 0.032 m faecium (T); X72865 (99.4%) OTU0580 Firmicutes Clostridia Clostridiales Veillonellaceae Dialister Dialister pneumosintes; 0.0007 0.0250 0.001 Z36297 (100.0%) OTU1032 Firmicutes Clostridia Clostridiales Peptostreptococcacea Peptostreptococcus Peptostreptococcus 0 0.2177 1.42E-07 e stomatis; GQ422715 (100.0%) OTU0343 Firmicutes Clostridia Clostridiales Peptostreptococcacea Peptostreptococcaceae 0.0014 0.0341 0.016 e bacterium oral taxon 091; GU400652 (97.1%) OTU0783 Firmicutes Clostridia Clostridiales Incertae Sedis XI Parvimonas Parvimonas micra (T); 0.0014 0.1092 3.09E-04 ATCC 33270; AY323523 (100.0%) OTU1073 Firmicutes Clostridia Clostridiales Incertae Sedis XI Anaerococcus Anaerococcus 0.0000 0.0129 0.006 lactolyticus (T); CCUG 31351; AF542233 Anaerococcus murdochii; DQ911243 (100.0%) OTU0473 Firmicutes Clostridia Clostridiales Clostridiaceae 0.0068 0 0.008 bacterium SH021; AB298756 (96.4%) OTU0748 Firmicutes Clostridia Clostridiales Lachnospiraceae Dorea Dorea longicatena (T); 0.0075 0.0569 0.544 III-35; AJ132842 (99.3%) OTU1040 Firmicutes Clostridia Clostridiales Lachnospiraceae Dorea Dorea longicatena (T); 0 0.0046 0.012 III-35; AJ132842 (99.3%) OTU0007 Firmicutes Clostridia Clostridiales Lachnospiraceae Lachnospira 1.4777 0.2913 9.23E-05 pectinoschiza; AY169414 (100.0%) OTU4020 Firmicutes Clostridia Clostridiales Lachnospiraceae Lachnospira 0.0041 0 0.023 pectinoschiza; AY169414 (98.6%) OTU0614 Firmicutes Clostridia Clostridiales Lachnospiraceae Lachnospira 0.0245 0.0038 0.006 pectinoschiza; AY169414 (99.3%) OTU0599 Firmicutes Clostridia Clostridiales Lachnospiraceae Lachnospira 0.0109 0.0008 0.006 pectinoschiza; AY169414 (99.3%) OTU0067 Firmicutes Clostridia Clostridiales Lachnospiraceae butyrate-producing 0.3786 0.0614 0.019 bacterium A2-166; AJ270489 (100.0%) OTU0457 Firmicutes Clostridia Clostridiales Lachnospiraceae Roseburia Roseburia intestinalis 0.0654 0.0152 0.278 (T); L1-82; AJ312385; Roseburia inulinivorans (T); A2- 194; AJ270473 (99.3%) OTU1704 Firmicutes Clostridia Clostridiales Lachnospiraceae Roseburia Roseburia intestinalis 0.0109 0.0030 0.024 (T); L1-82; AJ312385; Roseburia inulinivorans (T); A2- 194; AJ270473 (97.8%) OTU1443 Firmicutes Clostridia Clostridiales Lachnospiraceae Ruminococcus torques; 0.0082 0.0008 0.009 FP929055 (97.5%) OTU0662 Firmicutes Clostridia Clostridiales Lachnospiraceae Roseburia Roseburia intestinalis 0.0129 0.0008 0.001 (T); L1-82; AJ312385 Roseburia inulinivorans (T); A2- 194; AJ270473 (99.3%) OTU1167 Firmicutes Clostridia Clostridiales Lachnospiraceae Roseburia Roseburia intestinalis 0.0163 0.0038 0.002 (T); L1-82; AJ312385 Roseburia inulinivorans (T); A2- 194; AJ270473 (96.5%) OTU0756 Firmicutes Clostridia Clostridiales Lachnospiraceae Hespellia porcina (T); 0.0163 0.0038 0.038 PPC80; AF445239; Robinsoniella peoriensis (T); PPC31; AF445285 (99.3%) OTU1036 Firmicutes Clostridia Clostridiales Incertae Sedis XIV Blautia Ruminococcus obeum; 0.0007 0.0068 0.027 X85101 (99.3%) OTU3710 Firmicutes Clostridia Clostridiales Blautia schinkii; 0.0007 0.0061 0.006 X94964 Blautia luti (T); bln9; AJ133124 Blautia hansenii; AB534168 Ruminococcus sp.; GU797893 (97.1%) OTU0916 Firmicutes Clostridia Clostridiales Lachnospiraceae Clostridium 0.0095 0.0455 0.006 symbiosum; M59112 Clostridium bolteae; AJ508452 Clostridium asparagiforme (T); N6; AJ582080 Clostridium clostridioforme (T); M59089 Clostridium aldenense (T); RMA 9741; DQ279736 (98.6%) OTU0911 Firmicutes Clostridia Clostridiales Lachnospiraceae Clostridium 0.0048 0.0303 9.05E-04 symbiosum; M59112 Clostridium bolteae; AJ508452 Clostridium asparagiforme (T); N6; AJ582080 Clostridium clostridioforme (T); M59089 (99.3%) # Only taxonomical assignments with an estimated confidence above 80% are shown. * If more than one bacterial strains share the same similarity with the OTU, all of them are listed. Table S6. Results of real-time qPCR Healthy p CRC patients volunteers value

Log of total 16S rRNA gene copy number per ng 8.15, 7.55, 5.82- - DNA* 6.53-8.45 7.79 Log of Bacteriodes spp. 16S rRNA gene copy 7.40, 6.54, 0-7.22 - number per ng DNA 4.03-7.88 Log of butyryl-CoA CoA transferase gene copy 6.59, 1.74, 0-6.62 - number per ng DNA 4.64-7.39 Amount of Bacteriodes spp. related to total bacteria 17.11, 10.89, 0- 0.005 (%) 3.98e-3 -55.35 86.01 Amount of butyrate producing bacteria related to 3.06, 2.26, 1.5e-4 total bacteria (%) 0.03-11.4 0-8.73 * Data are shown as median, range.

References of supplementary materials Li M, Zhou H, Hua W, Wang B, Wang S, Zhao G, et al. (2009). Molecular diversity of Bacteroides spp. in human fecal microbiota as determined by group-specific 16s rRNA gene clone library analysis. Syst Appl Microbiol 32(3): 193-200.

Louis P, Flint HJ. (2007). Development of a semiquantitative degenerate real-time PCR-based assay for estimation of numbers of butyryl-coenzyme A (CoA) CoA transferase genes in complex bacterial samples. Appl Environ Microbiol 73(6): 2009-2012.

Nadkarni MA, Martin FE, Jacques NA, Hunter N. (2002). Determination of bacterial load by real- time PCR using a broad-range (universal) probe and primers set. Microbiology 148(Pt 1): 257- 266.