Plate 2 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: West Thurrock (WT1: P1 tag GTGAG; WT2: P2 tag TACGC; WT3: P3 tag GTCAC), Purfleet (P1: P4 tag CTACT; P2: P5 tag TCTGT; P3: P6 tag GATGA), Crossness (XN1: P7 tag TGACT; XN2: P8 tag ATGCT; XN3: P9 tag TCGTC), Beckton (B1: P10 tag TATAC; B2: P11 tag ACGAC; B3: P12 tag TCGCT), Woolwich (WW1: P13 tag TACAT; WW2: P14 tag GACGT; WW3: P15 tag TACTA), London Bridge (LB1: P16 tag ACTAT; LB2: P17 tag AGTGT; LB3: P18 tag ATAGT), Teddington (T1: P19 tag ACGCA; T2: P20 tag AGACA; T3: P21 tag ATCGA).