title: Human colorectal mucosa-associated bacterial 16S rDNA
organism: Bacteria
description: The biopsies were obtained from 5 male and 5 female healthy subjects by colonoscopy. The recruitment criteria were 45-70 years of age, non-smokers. All subjects had one biopsy taken using the same standard procedure from each of the following locations; caecum, transverse colon, sigmoid colon and rectum. The biopsies were snap frozen in liquid N2 and stored at -80°C prior to analysis.   Total DNA was extracted and purified from the biopsies using the AllPrep DNA/RNA Mini Kit, according to the manufacturer’s instructions. Subsamples of each DNA extract was used as the template for PCR-based amplification of the bacterial V1-V3 region of the rrs-genes using a barcoded pyrotagging approach. To avoid potential problems caused by the large quantities of human DNA present in the samples a nested approach was followed for the amplification of the Bacteria rrs-genes. First, 100-500 ng of total DNA extracted from each biopsy sample were used for the PCR amplification of the nearly full length 16S rRNA genes using primers 27f and 1492r. Then, 100 ng of the resulting purified products were used as templates for a second round of amplifications. The V1-V3 region of the genes were amplified using fusion primers containing the Roche-454 sequencing adapters followed by a small linker segment and the 5’ end of primers 8f (AGAGTTTGATCCTGG) and 515r (TTACCGCGGCTGCT). The reverse primer carried a short barcode sequence after the linker sequence. The amplification procedure consisted of 25 cycles of 30 s at 94ºC, 30 s at 55ºC, and 30 s at 72ºC. All samples were amplified in triplicate (hence three different barcodes/reactions per sample). The quality of the resulting products was checked on agarose gels, and then equal amounts of each product were pooled and cleaned by gel extraction using the QIAquick Gel Extraction Kit (QIAGEN). The resulting product was checked again by agarose electrophoresis, its concentration measured using a ND-1000 spectrophotometer (NanoDrop Technologies), and then sequenced using a Roche 454 GS FLX sequencer with titanium chemistry.    A description of barcodes used for each sample, and sample origin follows:    #SampleID BarcodeSequence LinkerPrimerSequence Sample Patient GIregion Sex Name Description  1a ACGAGTGCGT  TTACCGCGGCTGCT 1 CG569 S female 69Sf 1  2a ACGCTCGACA  TTACCGCGGCTGCT 2 CG550 S male 50Sm 2  3a AGACGCACTC  TTACCGCGGCTGCT 3 CG566 C male 66Cm 3  4a AGCACTGTAG  TTACCGCGGCTGCT 4 F001 S male 01Sm 4  5a ATCAGACACG  TTACCGCGGCTGCT 5 CG542 T female 42Tf 5  6a ATATCGCGAG  TTACCGCGGCTGCT 6 CG569 T female 69Tf 6  7a CGTGTCTCTA  TTACCGCGGCTGCT 7 CG517 C female 17Cf 7  8a CTCGCGTGTC  TTACCGCGGCTGCT 8 CG563 C male 63Cm 8  9a TGTGCGCGTG  TTACCGCGGCTGCT 9 CG507 C male 07Cm 9  10a TCTCTATGCG  TTACCGCGGCTGCT 10 CG507 T male 07Tm 10  11a TGATACGTCT  TTACCGCGGCTGCT 11 CG544 R female 44Rf 11  12a TGTATCACAG  TTACCGCGGCTGCT 12 CG541 C female 41Cf 12  13a CATAGTAGTG  TTACCGCGGCTGCT 13 CG517 R female 17Rf 13  14a CGAGAGATAC  TTACCGCGGCTGCT 14 CG544 C female 44Cf 14  15a ATACGACGTA  TTACCGCGGCTGCT 15 CG563 S male 63Sm 15  16a TCACGTACTA  TTACCGCGGCTGCT 16 CG542 S female 42Sf 16  17a CGTCTAGTAC  TTACCGCGGCTGCT 17 CG542 R female 42Rf 17  18a TCTACGTAGC  TTACCGCGGCTGCT 18 CG541 R female 41Rf 18  19a TGTACTACTC  TTACCGCGGCTGCT 19 CG544 T female 44Tf 19  20a ACGACTACAG  TTACCGCGGCTGCT 20 CG550 R male 50Rm 20  21a CGTAGACTAG  TTACCGCGGCTGCT 21 CG566 S male 66Sm 21  22a TACGAGTATG  TTACCGCGGCTGCT 22 CG569 C female 69Cf 22  23a TACTCTCGTG  TTACCGCGGCTGCT 23 CG563 T male 63Tm 23  24a TAGAGACGAG  TTACCGCGGCTGCT 24 CG563 R male 63Rm 24  25a TGTCGTCGCA  TTACCGCGGCTGCT 25 CG541 T female 41Tf 25  26a ACACATACGC  TTACCGCGGCTGCT 26 CG541 S female 41Sf 26  28a ACATGACGAC  TTACCGCGGCTGCT 28 CG507 S male 07Sm 28  29a ACGACAGCTC  TTACCGCGGCTGCT 29 CG550 T male 50Tm 29  30a ACGTCTCATC  TTACCGCGGCTGCT 30 CG566 R male 66Rm 30  31a ACTCATCTAC  TTACCGCGGCTGCT 31 CG542 C female 42Cf 31  32a ACTCGCGCAC  TTACCGCGGCTGCT 32 CG517 T female 17Tf 32  33a AGAGCGTCAC  TTACCGCGGCTGCT 33 CG550 C male 50Cm 33  34a AGCGACTAGC  TTACCGCGGCTGCT 34 CG566 T male 66Tm 34  35a AGTAGTGATC  TTACCGCGGCTGCT 35 CG544 S female 44Sf 35  36a AGTGACACAC  TTACCGCGGCTGCT 36 CG517 S female 17Sf 36  37a AGTGTATGTC  TTACCGCGGCTGCT 37 CG569 R female 69Rf 37  38a ATAGATAGAC  TTACCGCGGCTGCT 38 F001 R male 01Rm 38  40a ATCTACTGAC  TTACCGCGGCTGCT 40 CG507 R male 07Rm 40  1b AACCAACC TTACCGCGGCTGCT 1 CG569 S female 69Sf 41  2b AACGAACG TTACCGCGGCTGCT 2 CG550 S male 50Sm 42  3b AACGTTCG TTACCGCGGCTGCT 3 CG566 C male 66Cm 43  4b AAGCTACC TTACCGCGGCTGCT 4 F001 S male 01Sm 44  5b AAGGTACG TTACCGCGGCTGCT 5 CG542 T female 42Tf 45  6b AATTGCGC TTACCGCGGCTGCT 6 CG569 T female 69Tf 46  7b ACACGTCA TTACCGCGGCTGCT 7 CG517 C female 17Cf 47  8b ACAGCTGT TTACCGCGGCTGCT 8 CG563 C male 63Cm 48  9b AACCAAGG TTACCGCGGCTGCT 9 CG507 C male 07Cm 49  10b AACGAAGC TTACCGCGGCTGCT 10 CG507 T male 07Tm 50  11b AACGTTGC TTACCGCGGCTGCT 11 CG544 R female 44Rf 51  12b AAGCTAGG TTACCGCGGCTGCT 12 CG541 C female 41Cf 52  13b AAGGTAGC TTACCGCGGCTGCT 13 CG517 R female 17Rf 53  14b AATTGGCC TTACCGCGGCTGCT 14 CG544 C female 44Cf 54  15b ACACGTGT TTACCGCGGCTGCT 15 CG563 S male 63Sm 55  16b ACAGGACA TTACCGCGGCTGCT 16 CG542 S female 42Sf 56  17b AACCATCG TTACCGCGGCTGCT 17 CG542 R female 42Rf 57  18b AACGATCC TTACCGCGGCTGCT 18 CG541 R female 41Rf 58  19b AAGCAACG TTACCGCGGCTGCT 19 CG544 T female 44Tf 59  20b AAGCTTCG TTACCGCGGCTGCT 20 CG550 R male 50Rm 60  21b AAGGTTCC TTACCGCGGCTGCT 21 CG566 S male 66Sm 61  22b ACACACAC TTACCGCGGCTGCT 22 CG569 C female 69Cf 62  23b ACACTCAG TTACCGCGGCTGCT 23 CG563 T male 63Tm 63  24b ACAGGAGT TTACCGCGGCTGCT 24 CG563 R male 63Rm 64  25b AACCATGC TTACCGCGGCTGCT 25 CG541 T female 41Tf 65  26b AACGATGG TTACCGCGGCTGCT 26 CG541 S female 41Sf 66  27b AAGCAAGC TTACCGCGGCTGCT 27 F001 T male 01Tm 67  28b AAGCTTGC TTACCGCGGCTGCT 28 CG507 S male 07Sm 68  29b AAGGTTGG TTACCGCGGCTGCT 29 CG550 T male 50Tm 69  30b ACACACTG TTACCGCGGCTGCT 30 CG566 R male 66Rm 70  31b ACACTCTC TTACCGCGGCTGCT 31 CG542 C female 42Cf 71  32b ACAGGTCT TTACCGCGGCTGCT 32 CG517 T female 17Tf 72  33b AACCGCAT TTACCGCGGCTGCT 33 CG550 C male 50Cm 73  34b AACGCCAT TTACCGCGGCTGCT 34 CG566 T male 66Tm 74  35b AAGCATCC TTACCGCGGCTGCT 35 CG544 S female 44Sf 75  36b AAGGAACC TTACCGCGGCTGCT 36 CG517 S female 17Sf 76  37b AATACCGC TTACCGCGGCTGCT 37 CG569 R female 69Rf 77  38b ACACAGAG TTACCGCGGCTGCT 38 F001 R male 01Rm 78  39b ACACTGAC TTACCGCGGCTGCT 39 F001 C male 01Cm 79  40b ACAGGTGA TTACCGCGGCTGCT 40 CG507 R male 07Rm 80  1c AACCGGTT TTACCGCGGCTGCT 1 CG569 S female 69Sf 81  2c AACGCGTT TTACCGCGGCTGCT 2 CG550 S male 50Sm 82  4c AAGGATGC TTACCGCGGCTGCT 4 F001 S male 01Sm 84  5c AATAGGCG TTACCGCGGCTGCT 5 CG542 T female 42Tf 85  6c ACACCAGT TTACCGCGGCTGCT 6 CG569 T female 69Tf 86  7c ACAGACTC TTACCGCGGCTGCT 7 CG517 C female 17Cf 87  8c ACAGTGAG TTACCGCGGCTGCT 8 CG563 C male 63Cm 88  11c AAGCGCAA TTACCGCGGCTGCT 11 CG544 R female 44Rf 91  12c AAGGCCAA TTACCGCGGCTGCT 12 CG541 C female 41Cf 92  13c AATTCCGG TTACCGCGGCTGCT 13 CG517 R female 17Rf 93  14c ACACCTCT TTACCGCGGCTGCT 14 CG544 C female 44Cf 94  15c ACAGAGAC TTACCGCGGCTGCT 15 CG563 S male 63Sm 95  16c ACAGTGTC TTACCGCGGCTGCT 16 CG542 S female 42Sf 96  17c AACCTAGC TTACCGCGGCTGCT 17 CG542 R female 42Rf 97  18c AACGGCTT TTACCGCGGCTGCT 18 CG541 R female 41Rf 98  19c AAGCGCTT TTACCGCGGCTGCT 19 CG544 T female 44Tf 99  20c AAGGCCTT TTACCGCGGCTGCT 20 CG550 R male 50Rm 100  21c AATTCGCG TTACCGCGGCTGCT 21 CG566 S male 66Sm 101  22c ACACCTGA TTACCGCGGCTGCT 22 CG569 C female 69Cf 102  23c ACAGAGTG TTACCGCGGCTGCT 23 CG563 T male 63Tm 103  24c ACCAACCA TTACCGCGGCTGCT 24 CG563 R male 63Rm 104  25c AACCTTCC TTACCGCGGCTGCT 25 CG541 T female 41Tf 105  26c AACGTACC TTACCGCGGCTGCT 26 CG541 S female 41Sf 106  27c AAGCGGAT TTACCGCGGCTGCT 27 F001 T male 01Tm 107  28c AAGGCGAT TTACCGCGGCTGCT 28 CG507 S male 07Sm 108  29c AATTCGGC TTACCGCGGCTGCT 29 CG550 T male 50Tm 109  30c ACACGACT TTACCGCGGCTGCT 30 CG566 R male 66Rm 110  32c ACCAACGT TTACCGCGGCTGCT 32 CG517 T female 17Tf 112  33c AACCTTGG TTACCGCGGCTGCT 33 CG550 C male 50Cm 113  34c AACGTAGG TTACCGCGGCTGCT 34 CG566 T male 66Tm 114  35c AAGCGGTA TTACCGCGGCTGCT 35 CG544 S female 44Sf 115  36c AAGGCGTA TTACCGCGGCTGCT 36 CG517 S female 17Sf 116  37c AATTGCCG TTACCGCGGCTGCT 37 CG569 R female 69Rf 117  38c ACACGAGA TTACCGCGGCTGCT 38 F001 R male 01Rm 118  39c ACAGCAGA TTACCGCGGCTGCT 39 F001 C male 01Cm 119  40c TTGGTTGG TTACCGCGGCTGCT 40 CG507 R male 07Rm 120