title: Changes in structure and function of bacterial communities during coconut leaf vermicomposting
design: The study focussed on four characteristic stages during the vermicomposting of coconut-leaf. Vermicomposting was carried out as a batch system in cement tanks. The tanks were covered with a slanting roof made of thatched coconut leaves to prevent sunlight and rainwater from falling directly into them.  Two cement tanks, 9 m long x 2 m wide x 1 m deep, subdivided into three 3-meter long compartments by brick and mortar walls were used for this study. Samples were collected from the two tanks (VA and VB) in triplicate (one from each compartment) during all four stages of the coconut leaf vermicomposting process. Specifically, i) on the 15th day upon completion of pre-decomposition stage just before the addition of earthworms (VA15 and VB15); ii) on 45th day, 30 days after addition of earthworms during the second stage (VA45 and VB45); iii) on 75th day, 60 days after addition of the earthworms when conversion of more than 50% substrates to vermicompost was complete (VA75 and VB75); and iv) on 105th day, 90 days after inoculation of the worms when mature, sieved coconut leaf vermicompost was ready (VA105 and VB105). In addition, 20 adult earthworms were collected from randomly chosen places and depths in each compartment during the 75th (EA75 and EB75) and 105th days (EA105 and EB105).    Total community DNA was extracted from 0.5 g of vermicompost using the MoBio Power Soil DNA kit (MoBio, Carlsbad, USA).   For pyrosequencing, partial bacterial 16S rRNA gene fragments were PCR-amplified in 25-cycle reactions using primer 515R-NK linked to the 454 Life Science’s FLX-A sequencing adaptor, and a sevenfold-degenerate primer 27F-YM+3 linked to the FLX-B sequencing adaptor. An 8-nt sample-specific barcode was added between the A-adaptor and primer 515R-NK and was used to bioinformatically separate amplicons from the multiplexed reactions post-sequencing.       In addition, a set of six controls prepared from three cloned 16S rRNA gene fragments of different GC content were used in the sequencing run. These control sequences were later used to optimize the sequence analysis pipeline.  Forward Sequecing primer: CCGCNGCKGCTGGCAC.   The table below describes the barcodes for the samples.     Sample   Barcode Library  CTRL ACGAACCT Single Control 1, Actinobacteria  CTRL ACGAACGA Single Control 2, Gammaproteobacteria  CTRL ACGAAGGT Single Control 3, Firmicutes  CTRL ACGACATC Mixed Control 4, A:G:F::1:1:1  CTRL ACGACTAC Mixed Control 5, A:G:F::1:2:3  CTRL ACGAGAAC Mixed Control 6, A:G:F::3:2:1  VA15 ACGAGTTC rep 1  VA15 ACGATGGA rep 2  VA15 ACTCACTC rep 3  VB15 ACTCCTCA rep 1  VB15 ACTCCTGT rep 2  VB15 ACTCGTCT rep 3  VA45 ACTCTCAC rep 1  VA45 ACTCTGTC rep 2  VA45 ACTGACAC rep 3  VB45 ACTGGAGA rep 1  VB45 ACTGTCTC rep 2  VB45 AGACACTC rep 3  VA75 AGACCTCA rep 1  VA75 AGACGACA rep 2  VA75 AGACTCAC rep 3  VB75 AGAGACAC rep 1  VB75 AGAGAGTC rep 2  VB75 AGAGGAGA rep 3  EA75 AGGAAGGA rep 1  EA75 AGGACAAC rep 2  EA75 AGGTAGGT rep 3  EB75 AGGTCATC rep 1  EB75 AGGTTCGT rep 2  EB75 AGTCACAC rep 3  VA105 AGTCCTCT rep 1  VA105 AGTCGTCA rep 2  VA105 AGTCTCTC rep 3  VB105 ATCCGCAA rep 1  VB105 ATCCGCTT rep 2  VB105 ATCCTACC rep 3  EA105 ATCGTTCC rep 1  EA105 CAACTCCT rep 2  EA105 CAACTGGT rep 3  EB105 CAAGACCT rep 1  EB105 CAAGACGA rep 2  EB105 CAAGAGGT rep 3  Each DNA was separately amplified in a 25 μl PCR reaction that contained 1.25 μl (20-50 ng) of DNA, 12.5 pmol of each primer, and 22.5 μl of Platinum® PCR SuperMix High Fidelity (Invitrogen). Reaction conditions included an initial denaturation at 95°C for 3 min followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 68°C for 1 min; and a final elongation at 68°C for 4 min on a Veriti® 96-well Thermal Cycler (Applied Biosystems). The PCR products were electrophoresed on 1% agarose gel in 0.5x TBE, stained with SYBR® Green I nucleic acid gel stain (Invitrogen) and scanned using Typhoon Trio+ Variable mode imager (GE Healthcare). The resultant images were quantified using Image Quant 5.2 (Molecular Dynamics). PCR products from replicates of each soil sample were then pooled in equimolar concentrations, followed by gel elution using Zymoclean™ Gel DNA Recovery Kit (Zymo Research). The eluted amplicons were quantified on the Experion System (Bio-Rad), followed by mixing of amplicons from all soil ages in equimolar concentration. The final mixed amplicon pool was further purified using the Agencourt AMPure XP system (Beckman Coulter Genomics) and submitted to the Georgia Genomics Facility at the University of Georgia (Athens, USA) for pyrosequencing. Emulsion PCRs were carried out as per their protocols.