Plate 1 Second-half
Community DNA extraction, PCR amplification of the 18S nuclear small subunit (nSSU) rDNA gene and 454 Roche metagenetic library preparation using the highly conservative metagenetic primers SSU_F_04 and SSU_R_22. The nSSU gene region was PCR amplified using independently MID-tagged fusion primers from community DNA, visualized by gel electrophoresis and purified, quantified on an Agilent bioanalyser and pooled in equimolar quantities. The purified amplicons pools were then sequenced in a single direction (A-Amplicon), using a 454 Roche GSFLX (454 Life Sciences, Roche Applied Science) sequencing platform at Liverpool University’s Centre for Genomic Research, UK.  Below are given the samples location, as well as the MID-tag used for PCR amplification.  Thames estuary samples: South Bank Centre (SBC3: P1 tag GTGAG), Cadogan Pier (CP1: P5 tag TCTGT; CP2: P6 tag GATGA; CP3: P7 tag TGACT), Old Isleworth (OI1: P8 tag ATGCT; OI2: P9 tag TCGTC, OI3: P10 tag TATAC), Hammersmith Bridge (HB1: P11 tag ACGAC; HB2: P12 tag TCGCT, HB3: P13 tag TACAT).    Mersey estuary samples: Rock Ferry (RF1: P2 tag TACGC; RF2: P3 tag GTCAC; RF3: P4 tag CTACT), Runcorn (RC1: P14 tag GACGT; RC2: P15 tag TACTA, RC3: P16 tag ACTAT), Fiddler's Ferry (FF1: P17 tag AGTGT; FF2: P18 tag ATAGT; FF3: P19 tag ACGCA), Cuerdley Marsh (CM1: P20 tag AGACA; CM2: P21 tag ATCGA, CM3: P22 tag TAGCA), Mersey Tunnel (MT1: P23 tag TGATA; MT2: P24 tag TGTCA; MT3: P25 tag GACTC), Eastham Ferry (EF1: P26 tag GAGAC; EF2: P27 tag TGCAC; EF3: P28 tag GCATA).