SRX285771 BPS IncP-1 1.1 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428737 trfA_Sampling1.1 tag B-1 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285772 BPS IncP-1 1.2 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428738 trfA_Sampling1.2 tag B-2 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285773 BPS IncP-1 1.3 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428739 trfA_Sampling1.3 tag B-3 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285774 BPS IncP-1 1.4 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428740 trfA_Sampling1.4 tag B-4 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285775 BPS IncP-1 2.1 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428741 trfA_Sampling2.1 tag B-5 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285776 BPS IncP-1 2.2 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428742 trfA_Sampling2.2 tag B-6 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285777 BPS IncP-1 2.3 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428743 trfA_Sampling2.3 tag B-7 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285779 BPS IncP-1 2.4 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428745 trfA_Sampling2.4 tag B-8 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285780 BPS IncP-1 3.1 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428746 trfA_Sampling3.1 tag B-9 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285782 BPS IncP-1 3.2 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428748 trfA_Sampling3.2 tag B-10 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285784 BPS IncP-1 3.3 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428750 trfA_Sampling3.3 tag B-11 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285785 BPS IncP-1 3.4 SRP020538 Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene SRS428751 trfA_Sampling3.4 tag B-12 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285990 BPS IncP-1 1.1 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428737 trfA_Sampling1.1 tag B-1 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285991 BPS IncP-1 1.1 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428737 trfA_Sampling1.1 tag B-1 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285992 BPS IncP-1 1.2 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428738 trfA_Sampling1.2 tag B-2 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285993 BPS IncP-1 1.2 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428738 trfA_Sampling1.2 tag B-2 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285994 BPS IncP-1 1.3 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428739 trfA_Sampling1.3 tag B-3 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285995 BPS IncP-1 1.3 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428739 trfA_Sampling1.3 tag B-3 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285996 BPS IncP-1 1.4 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428740 trfA_Sampling1.4 tag B-4 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285997 BPS IncP-1 1.4 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428740 trfA_Sampling1.4 tag B-4 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285998 BPS IncP-1 2.1 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428741 trfA_Sampling2.1 tag B-5 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX285999 BPS IncP-1 2.1 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428741 trfA_Sampling2.1 tag B-5 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286000 BPS IncP-1 2.2 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428742 trfA_Sampling2.2 tag B-6 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286001 BPS IncP-1 2.2 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428742 trfA_Sampling2.2 tag B-6 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286002 BPS IncP-1 2.3 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428743 trfA_Sampling2.3 tag B-7 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286003 BPS IncP-1 2.3 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428743 trfA_Sampling2.3 tag B-7 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286004 BPS IncP-1 2.4 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428745 trfA_Sampling2.4 tag B-8 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286005 BPS IncP-1 2.4 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428745 trfA_Sampling2.4 tag B-8 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286006 BPS IncP-1 3.1 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428746 trfA_Sampling3.1 tag B-9 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286007 BPS IncP-1 3.1 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428746 trfA_Sampling3.1 tag B-9 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286008 BPS IncP-1 3.2 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428748 trfA_Sampling3.2 tag B-10 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286009 BPS IncP-1 3.2 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428748 trfA_Sampling3.2 tag B-10 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286010 BPS IncP-1 3.3 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428750 trfA_Sampling3.3 tag B-11 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286011 BPS IncP-1 3.3 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428750 trfA_Sampling3.3 tag B-11 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286012 BPS IncP-1 3.4 d SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428751 trfA_Sampling3.4 tag B-12 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium SRX286013 BPS IncP-1 3.4 g SRP020538 "Pyrosequencing of the IncP-1 trfA genes was performed on TC-DNA from all replicas at all three sampling times, 12 samples in total. A 281 bp region of the IncP-1 trfA gene was amplified using the three primer sets previously described by Bahl et al. (Bahl et al., 2009) that target either the IncP-1α, β and ε supgroups, IncP-1γ or IncP-1δ. Primes had U-linkers added (Holmsgaard et al., in prep). PCR amplification was done by adding 1 µl DNA to a 25 µl reactions mixture of 1x Phusion HF buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mixture, 0.02 U/µl Phusion Hot Start DNA Polymerase (Finnzymes), 0.4 µM of each primer (Sigma-Aldrich, St. Louis, Mo, USA). PCR conditions were a denaturation at 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 67°C for 30 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. After PCR amplification, the samples were held at 70 °C for 3 min and then placed on ice until the products were analyzed on 1% (w/v) agarose gel with ethidium bromide and visualized with UV-Illumination. Bands of the PCR products were cut from the gel and purified by the Montage Gel extraction kit (Millipore, Billerica, MA, USA). A second PCR amplification was performed as described above, except that primers with a 454 Titanium adapter, 10 nucleotide barcodes and U-linkers were used and with an annealing temperature of 58°C for the U-linker. Furthermore, PCR cycles were reduced to 15. PCR products were again analysed by agarose gel electrophoresis and purified from the gel. The amplicons with adapters and barcodes were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA Sequencing was done as part of a larger run in one of two regions on a GS FLX Titanium PicoTiterPlate using the GS FLX pyrosequencing system (Roche, Basel, Switzerland). Bahl MI, Burmølle M, Meisner A, Hansen LH & Sørensen SJ (2009) All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid 62: 134-139. Holmsgaard PN, Hansen LH, Sørensen SJ (in prep.) Simultaneous pyrosequencing of the 16S rRNA gene, IncP-1 trfA gene and merA gene" SRS428751 trfA_Sampling3.4 tag B-12 AMPLICON METAGENOMIC PCR 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX Titanium