Plate 1 First half-plate
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: Shoeburyness East (SNE1: P1 tag GTGAG; SNE2: P2 tag TACGC; SNE3: P3 tag GTCAC), Southend on Sea (SE1: P4 tag CTACT; SE2: P5 tag TCTGT; SE3: P6 tag GATGA), Greenwich (GW1: P16 tag ACTAT; GW2: P17 tag AGTGT; GW3: P18 tag ATAGT), Kew (K1: P25 tag GACTC; K2: P26 tag GAGAC; K3: P27 tag TGCAC), South Bank Centre (SBC2: P28 tag GCATA).    Mersey estuary samples: The Narrows (TN1: P7 tag TGACT; TN2: P8 tag ATGCT; TN3: P9 tag TCGTC), Hale Head Shore (HH1: P10 tag TATAC; HH2: P11 tag ACGAC; HH3: P12 tag TCGCT), Speke (SK1: P13 tag TACAT; SK2: P14 tag GACGT; SK3: P15 tag TACTA), Ellesmere Bank (EB1: P19 tag ACGCA; EB2: P20 tag AGACA; EB3: P21 tag ATCGA), Liverpool Airport (LA1: P22 tag TAGCA; LA2: P23 tag TGATA; LA3: P24 tag TGTCA).