SRX039396 16S rRNA gene Bacteria V3V4 amplification SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160632 JBL_J07_AT bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039397 16S rRNA gene Bacteria V3V4 amplification_BS SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160633 JBL_J07_BS bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039398 16S rRNA gene Bacteria V3V4 amplification_Dig SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160634 JBL_J07_Dig bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039399 16S rRNA gene Bacteria V3V4 amplification_GT SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160635 JBL_J07_GT bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039400 16S rRNA gene Bacteria V3V4 amplification_LBS SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160636 JBL_J07_LBS bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039401 16S rRNA gene Bacteria V3V4 amplification_BT SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160637 BT bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039402 16S rRNA gene Bacteria V3V4 amplification_SV SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160638 JBL_J07_SV bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039403 16S rRNA gene Bacteria V3V4 amplification_MS SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160639 JBL_J07_MS bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039404 16S rRNA gene Bacteria V3V4 amplification_TS SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160640 JBL_J07_TS bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039405 16S rRNA gene Bacteria V3V4 amplification_Holm SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160641 JBL_J07_Holm bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039406 16S rRNA gene Bacteria V3V4 amplification_Hag SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160642 JBL_J07_Hag bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039407 16S rRNA gene Bacteria V3V4 amplification_Hal SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160643 JBL_J07_Hal bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039408 16S rRNA gene Bacteria V3V4 amplification_VN SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160644 JBL_J07_VN bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX039409 GJQHPFX01.ON_0.sff SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS160645 JBL_J07_ON bv3v4 AMPLICON GENOMIC PCR 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX129174 16S rRNA gene Bacteria V3V4 amplification_HES SRP005457 JBLJ07 The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 (5’-CCTACGGGNGGCWGCAG-3’) and individually bar-coded reverse primers 805 (5’-GACTACHVGGGTATCTAATCC-3’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample. SRS300122 JBL_J07_HES bv3v4 AMPLICON GENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium