Plate 2 First-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: Allhallows (AH1: P13 tag TACAT; AH2: P14 tag GACGT; AH3: P15 tag TACTA), Canvey Island (CB1: P16 tag ACTAT; CB2: P17 tag AGTGT; CB3: P18 tag ATAGT), Stanford Le Hope (SLH1: P19 tag ACGCA; SLH2: P20 tag AGACA; SLH3: P21 tag ATCGA), Coalhouse Fort (CF1: P22 tag TAGCA; CF2: P23 tag TGATA; CF3: P24 tag TGTCA), Gravesend (GV1: P25 tag GACTC; GV2: P26 tag GAGAC; GV3: P27 tag TGCAC).    Mersey estuary samples: Egremont (EG1: P1 tag GTGAG; EG2: P2 tag TACGC; EG3: P3 tag GTCAC), East Hale (EH1: P4 tag CTACT; EH2: P5 tag TCTGT; EH3: P6 tag GATGA), Forest Way (FW1: P7 tag TGACT; FW2: P8 tag ATGCT; FW3: P9 tag TCGTC), Howley Weir (HW1: P10 tag TATAC; HW2: P11 tag ACGAC; HW3: P12 tag TCGCT).