Summary: the Supplementary Materials Include Fourfigures and Sixtables That Are Listed Below

Summary: The supplementary materials include fourfigures and sixtables that are listed below.

Figures

Figure S1:(a-c) Rarefaction curves of all samples (a), healthy control samples(b) and samples of CRCpatients(c); (d-f) Shannon diversity index curves of all samples (d), healthy control samples(e) and samples of CRCpatients(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 OTUsidentified at several different similarity levels. The x-axis shows theidentity, the y-axis shows the number of OTUs.

Figure S4:PCA scores plotsbased 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 healthyvolunteers; red circles represent CRC patients.

Tables

Table S1. Primers and amplification conditions for qPCR assays in this study

Target bacterial group / Primers / Primer Sequences / References / Amplification conditions
Total bacteria / Uni-1 (331F) / 5’-TCCTACGGGAGGCAGCAGT-3’ / (Nadkarni et al., 2002) / Initialdenaturing at 95°Cfor 4 min;40 cycles ofdenaturing at 95°C for 15 s, and annealing/extension at 60°C for 1 min.
Uni-2 (797R) / 5’-GGACTACCAGGGTATCTAATCCTGTT-3’
Bacteroidesspp. / Bfr-F / 5’-CTGAACCAGCCAAGTAGCG-3’ / (Li et al., 2009) / Initial denaturing at 95°Cfor 2 min;40 cycles ofdenaturing 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 producing bacteria / BCoATscrF / 5’-GCIGAICATTTCACITGGAAYWSITGGCAYATG-3’ / (Louis Flint, 2007) / Initial denaturing at 95°Cfor 3 min;40 cycles ofdenaturing at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 30 s.
BCoATscrR / 5’-CCTGCCTTTGCAATRTCIACRAANGC-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

Healthyvolunteers / CRC patients
Subject name / Total sequences / Unique sequences / OTUs (98%) / Subject name / Total sequences / Unique sequences / OTUs (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 asmean ±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 ofthe 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 number / Taxonomical assignments (RDP Classifier)# / Nearest neighboring bacterium/bacteria (similarity)* / Relative contribution (%) / p value
(Mann-Whitney test)
Phylum / Class / Order / Family / Genus / H / C
OTU0005 / Bacteroidetes / Bacteroidia / Bacteroidales / Bacteroidaceae / Bacteroides / Bacteroides vulgatus; AB510712 (100.0% ) / 7.3955 / 2.0929 / 1.93E-04
OTU0945 / Bacteroidetes / Bacteroidia / Bacteroidales / Bacteroidaceae / Bacteroides / Bacteroides vulgatus; AB510712 (98.7%) / 0.0327 / 0.0083 / 5.87E-04
OTU4160 / Bacteroidetes / Bacteroidia / Bacteroidales / Bacteroidaceae / Bacteroides / Bacteroides uniformis; AB510711 (97.5%) / 0.0041 / 0 / 0.023
OTU3045 / Bacteroidetes / Bacteroidia / Bacteroidales / Bacteroidaceae / Bacteroides / Bacteroides fragilis(T); ATCC 25285; CR626927 (98.1%) / 0 / 0.0114 / 0.006
OTU0185 / Bacteroidetes / Bacteroidia / Bacteroidales / Rikenellaceae / Alistipes / Alistipes indistinctus; AB490804 (97.5% ) / 0.0504 / 0.0053 / 3.82E-04
OTU0845 / Actinobacteria / Actinobacteria / Bifidobacteriales / Bifidobacteriaceae / Bifidobacterium / Bifidobacterium catenulatum (T); ATCC27539; AF432082
Bifidobacterium pseudocatenulatum;AY166521
Bifidobacterium thermacidophilum subsp. thermacidophilum; AY166524
Bifidobacterium angulatum; AB489093 (97.9%) / 0.0041 / 0.0303 / 3.40E-04
OTU1336 / Actinobacteria / Actinobacteria / Bifidobacterium pseudocatenulatum; AB125917
Bifidobacterium catenulatum(T); ATCC27539; AF432082
Bifidobacterium angulatum; AB489093 (95.1%) / 0.0007 / 0.0076 / 0.013
OTU0176 / Firmicutes / Clostridia / Clostridiales / Ruminococcaceae / Oscillibacter / Oscillospira guilliermondii; AB040499 (97.1% ) / 0.0443 / 0.0068 / 7.14E-04
OTU0039 / Proteobacteria / Gammaproteobacteria / Enterobacteriales / Enterobacteriaceae / Escherichia/Shigella / Shigella dysenteriae(T); X96966
Escherichia coli; X80731
Hafnia alvei; Z83203
Shigella sonnei; X80726 (100.0%) / 0.9398 / 2.8150 / 2.42E-05
OTU0484 / Proteobacteria / Gammaproteobacteria / Enterobacteriales / Enterobacteriaceae / Escherichia/Shigella / Hafnia alvei; Z83203
Escherichia albertii(T); LMG 20976; AJ508775
Shigella boydii;AY696670
Escherichia coli; CP000243 (99.4%) / 0 / 0.0061 / 0.006
OTU1955 / Proteobacteria / Gammaproteobacteria / Enterobacteriales / Enterobacteriaceae / Escherichia/Shigella / Escherichia coli;X80731
Hafnia alvei; Z83203 (98.8%) / 0.0007 / 0.0083 / 0.006
OTU2525 / Proteobacteria / Gammaproteobacteria / Enterobacteriales / Enterobacteriaceae / Klebsiella oxytoca; FM207542 (97.0%) / 0.0014 / 0.0121 / 0.010
OTU1203 / Proteobacteria / Gammaproteobacteria / Escherichia fergusonii; AJ277981 (97.000% similar) / 0.0041 / 0.0174 / 0.012
OTU0901 / Proteobacteria / Gammaproteobacteria / Pseudomonadales / Moraxellaceae / Acinetobacter / Acinetobacter johnsonii; DQ257432 (100.0%) / 0.0007 / 0.0091 / 0.012
OTU3671 / Proteobacteria / Gammaproteobacteria / Enterobacteriales / Enterobacteriaceae / Citrobacter freundii; FJ542329 (98.8%) / 0 / 0.0046 / 0.012
OTU1380 / Fusobacteria / Fusobacteria / Fusobacteriales / Fusobacteriaceae / Fusobacterium / Fusobacterium periodonticum; AJ810271; Fusobacterium nucleatum E9_12; AF481217 (100.0%) / 0.0020 / 0.0614 / 0.005
OTU1801 / Actinobacteria / Actinobacteria / Coriobacteriales / Coriobacteriaceae / Eggerthella / Eggerthella lenta(T); ATCC25559; AF292375 (97.9%) / 0.0014 / 0.0121 / 0.009
OTU0147 / Firmicutes / Clostridium spiroforme; X73441 (94.4%) / 0.8424 / 0.4377 / 0.009
OTU0391 / Firmicutes / Bacilli / Bacillales / Staphylococcaceae / Gemella / Gemella morbillorum; GU561406 (100.0%) / 0.0068 / 0.0728 / 0.006
OTU1407 / Firmicutes / Bacilli / Lactobacillales / Enterococcaceae / Enterococcus / Enterococcus ratti; AF326472
Enterococcus faecium; AB018210
Enterococcus faecalis; EU717961
Enterococcus durans; EU717964 (98.8%) / 0 / 0.0190 / 0.003
OTU2673 / Firmicutes / Bacilli / Lactobacillales / Enterococcaceae / Enterococcus / Enterococcus faecium(T); LMG 11423; AJ301830
Enterococcus durans(T); DSM20633; AJ276354
Enterococcus pseudoavium; AJ301837
Enterococcus hirae; AJ276356 (99.4%) / 0 / 0.0038 / 0.012
OTU1174 / Firmicutes / Clostridia / Clostridiales / Clostridium innocuum (T); M23732 (99.4%) / 0.0007 / 0.0228 / 0.005
OTU1435 / Firmicutes / Bacilli / Lactobacillales / Streptococcaceae / Streptococcus / Streptococcus thermophilus(T); ATCC 19258; AY188354 (99.3%) / 0.0007 / 0.0106 / 0.011
OTU1803 / Firmicutes / Bacilli / Lactobacillales / Streptococcaceae / Streptococcus / Streptococcus gallolyticus(T);LAB617; Z94012(97.6%) / 0 / 0.0053 / 0.012
OTU0432 / Firmicutes / Clostridia / Clostridiales / Veillonellaceae / Phascolarctobacterium / Phascolarctobacterium faecium(T); X72865 (99.4%) / 0.0259 / 0.0091 / 0.032
OTU0580 / Firmicutes / Clostridia / Clostridiales / Veillonellaceae / Dialister / Dialister pneumosintes; Z36297 (100.0%) / 0.0007 / 0.0250 / 0.001
OTU1032 / Firmicutes / Clostridia / Clostridiales / Peptostreptococcaceae / Peptostreptococcus / Peptostreptococcus stomatis; GQ422715 (100.0%) / 0 / 0.2177 / 1.42E-07
OTU0343 / Firmicutes / Clostridia / Clostridiales / Peptostreptococcaceae / Peptostreptococcaceae bacterium oral taxon 091; GU400652 (97.1%) / 0.0014 / 0.0341 / 0.016
OTU0783 / Firmicutes / Clostridia / Clostridiales / Incertae Sedis XI / Parvimonas / Parvimonas micra(T); ATCC 33270; AY323523 (100.0%) / 0.0014 / 0.1092 / 3.09E-04
OTU1073 / Firmicutes / Clostridia / Clostridiales / Incertae Sedis XI / Anaerococcus / Anaerococcus lactolyticus(T); CCUG 31351; AF542233
Anaerococcus murdochii; DQ911243 (100.0%) / 0.0000 / 0.0129 / 0.006
OTU0473 / Firmicutes / Clostridia / Clostridiales / Clostridiaceae bacterium SH021; AB298756 (96.4%) / 0.0068 / 0 / 0.008
OTU0748 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Dorea / Dorea longicatena(T); III-35; AJ132842 (99.3%) / 0.0075 / 0.0569 / 0.544
OTU1040 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Dorea / Dorea longicatena(T); III-35; AJ132842 (99.3%) / 0 / 0.0046 / 0.012
OTU0007 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Lachnospira pectinoschiza; AY169414 (100.0%) / 1.4777 / 0.2913 / 9.23E-05
OTU4020 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Lachnospira pectinoschiza; AY169414 (98.6%) / 0.0041 / 0 / 0.023
OTU0614 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Lachnospira pectinoschiza; AY169414 (99.3%) / 0.0245 / 0.0038 / 0.006
OTU0599 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Lachnospira pectinoschiza; AY169414 (99.3%) / 0.0109 / 0.0008 / 0.006
OTU0067 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / butyrate-producing bacterium A2-166; AJ270489 (100.0%) / 0.3786 / 0.0614 / 0.019
OTU0457 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Roseburia / Roseburia intestinalis(T); L1-82; AJ312385;
Roseburia inulinivorans(T); A2-194; AJ270473 (99.3%) / 0.0654 / 0.0152 / 0.278
OTU1704 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Roseburia / Roseburia intestinalis(T); L1-82; AJ312385;
Roseburia inulinivorans(T); A2-194; AJ270473(97.8%) / 0.0109 / 0.0030 / 0.024
OTU1443 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Ruminococcus torques; FP929055 (97.5%) / 0.0082 / 0.0008 / 0.009
OTU0662 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Roseburia / Roseburia intestinalis(T); L1-82; AJ312385
Roseburia inulinivorans(T); A2-194; AJ270473 (99.3%) / 0.0129 / 0.0008 / 0.001
OTU1167 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Roseburia / Roseburia intestinalis(T); L1-82; AJ312385
Roseburia inulinivorans(T); A2-194; AJ270473 (96.5%) / 0.0163 / 0.0038 / 0.002
OTU0756 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Hespellia porcina(T); PPC80; AF445239;
Robinsoniella peoriensis(T); PPC31; AF445285 (99.3%) / 0.0163 / 0.0038 / 0.038
OTU1036 / Firmicutes / Clostridia / Clostridiales / Incertae Sedis XIV / Blautia / Ruminococcus obeum; X85101 (99.3%) / 0.0007 / 0.0068 / 0.027
OTU3710 / Firmicutes / Clostridia / Clostridiales / Blautia schinkii; X94964
Blautia luti(T); bln9; AJ133124
Blautia hansenii; AB534168
Ruminococcus sp.; GU797893 (97.1%) / 0.0007 / 0.0061 / 0.006
OTU0916 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Clostridium symbiosum; M59112
Clostridium bolteae; AJ508452
Clostridium asparagiforme(T); N6; AJ582080
Clostridium clostridioforme(T); M59089
Clostridium aldenense(T); RMA 9741; DQ279736 (98.6%) / 0.0095 / 0.0455 / 0.006
OTU0911 / Firmicutes / Clostridia / Clostridiales / Lachnospiraceae / Clostridium symbiosum; M59112
Clostridium bolteae; AJ508452
Clostridium asparagiforme(T); N6; AJ582080
Clostridium clostridioforme(T); M59089 (99.3%) / 0.0048 / 0.0303 / 9.05E-04

# 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 volunteers / CRC patients / p value
Log of total 16S rRNA gene copy number per ng DNA* / 8.15,
6.53-8.45 / 7.55, 5.82-7.79 / -
Log of Bacteriodes spp. 16S rRNA gene copy number per ng DNA / 7.40,
4.03-7.88 / 6.54,0-7.22 / -
Log of butyryl-CoACoA transferase gene copy number per ng DNA / 6.59,
4.64-7.39 / 1.74, 0-6.62 / -
Amount ofBacteriodes spp. related to total bacteria (%) / 17.11,
3.98e-3 -55.35 / 10.89, 0-86.01 / 0.005
Amount of butyrate producing bacteria related to total bacteria (%) / 3.06,
0.03-11.4 / 2.26,
0-8.73 / 1.5e-4

* 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.