Sequencing of 16S rRNA ampicons from bacterioplankton of 4 Finnish Lakes
Epilimnion samples were pooled from 0, 1 and 2 m depths. Metalimnion samples were pooled from 3 and 4 m depths. Hypolimnion samples were from 5 m depth. Water was passed through a 50 µm sieve to remove larger zooplankton. Pooled water was transported to the laboratory and kept at +4 C ° until it was concentrated with tangential flow filtration (Durapore cassette, pore size 0.22 µm, Millipore) within 6 hours of sampling. Concentrated water samples were frozen and freeze dried with Alpha 1-4 LD plus (Christ, Osterode, Germany). DNA was extracted by phenol-chloroform extraction (Griffits, et al., 2000) from 0.4 mg of freeze-dried material. Amplification of bacterial 16S rRNA genes (E. coli positions 341 to 805) was conducted using general bacteria primers 341F (5’-CCTACGGGNGGCWGCAG-3’) and 805R (5’-GACTACHVGGGTATCTAATCC-3’) (Herlemann et al. 2011). PCR and amplicon processing prior to sequencing was performed as described in Eiler et al. (2011), except for purification  of PCR products  with Agencourt AMPure XP purification system (Beckman Coulter, Danvers, USA) and amplicon quantification with SYBR Green in a Qubit fluorometer (Invitrogen Corporation, Carlsbad, USA). Amplicons were sequenced with Titanium chemistry from adaptor A, using a 454 GS-FLX system (454 Life Sciences, Branford, CT) at the Institute of Biotechnology hosted by University of Helsinki, Finland. The resulting reads carried the sample-specific molecular barcode and covered the entire V4 region of the 16S rRNA gene as well as flanking regions.  Barcodes resemble samples as given:   barcode ATCAT vm_h_s      barcode ATCTC vm_m_s      barcode ATCGA vm_e_s      barcode ATGAC n_h_s      barcode ATGTG n_m_s      barcode ATGCA n_e_s      barcode ACATC vk_h_s      barcode ACACA vk_m_s      barcode TATCA vk_e_s      barcode TATGC mj_h_s      barcode TACAC mj_m_s      barcode TACTA mj_e_s      barcode TACGT vm_h_a      barcode TAGAG vm_m_a      barcode TAGTC vm_e_a      barcode TAGCT n_h_a      barcode CATAC n_m_a      barcode CATCT n_e_a      barcode CATGA vk_h_a      barcode CACAT vk_m_a      barcode CACTC vk_e_a      barcode CAGTA mj_h_a      barcode CAGCG mj_m_a      barcode CTATC mj_e_a
Uppsala University, Department of Ecology and Gene
GJVT7LS08