ITS-seq of fungi community: pH value 8.26: Sample 2012_ITS_II_pH1_9
Amplification of the ITS2 region, ITS primer pair ITS4-TAG reverse (5’TCCTCCGCTTATTGATATGC) carrying the barcode and gITS7 forward (5’GTGARTCATCGARTCTTTG).18S variable regions V5-V8 was done in a nested PCR approach. The first PCR, the primer pair NS1 forward (5’GTAGTCATATGCTTGTCTC) and NS8 reverse (5’TCCGCAGGTTCACCTACGGA). The second, nested PCR used the primer pair nu-SSU-0817 forward (5’TTAGCATGGAATAATRRAATAGGA) and nu-SSU-1536 reverse (5’ATTGCAATGCYCTATCCCCA).This was done with all samples and with the negative control.DNA samples deriving from the various replicates (5), incubation times (1-4 weeks) and pH treatments (in situ and 7.67) were individually amplified. One seawater sample to the time point of sampling was included as a positive control. For a multiplex sequencing approach, 42 different primers were constructed per markergene. Products were sequenced on a Roche 454 GS-FLX Titanium platform (LGC, Berlin, Germany). 