Generic sample from Caenorhabditis elegans
Caenorhabditis elegans
Forty-one nematode species (Table 2) representing most clades (Holterman et al. 2006) were hand-picked and placed in ZR BashingBead Lysis Tubes (Zymo Research Corp, Santa Ana, CA) for either single nematode amplification (single nematode per tube – sPCR) or multiple nematode amplification (all nematode species placed together – mPCR). Tubes were placed into the Mini-BeadBeater (BioSpec Products, Inc. Bartlesville, OK) and processed at max speed for 2 minutes. Genomic DNA was extracted using ZR Soil Microbe DNA kit according to the manufacture’s protocol.  Eluted DNA was stored at -20?C until used as PCR template.  The 18S and 28S barcoding regions were amplified using fusion primers that included a proprietary primer sequence (A or B) of the 454 GS and priming sequences of 18S and 28S. PCR amplifications of 18S were performed using 1 ?l genomic DNA template in 25 ?l reactions containing 2.5 ?l of 10X buffer with MgCL2, 0.5 ?l of each of dNTP, 0.5 ?l of primers A-NF1 (5’-GCCTCCCTCGCGCCATCAGGGTGGTGCATGGCCGTTCTTAGTT-3’) and B-18Sr2b (5’-GCCTTGCCAGCCCGCTCAGTACAAAGGGCAGGGACGTAAT-3’), and 0.165 ?l of DyNAzyme Hot Start DNA polymerase (BioLabs New England Inc. Ipswitch, MA). The thermal cycling involved denaturation at 94?C for 10 min, followed by 35 cycles of denaturation at 94?C for 1 min, annealing at 58?C for 30 s, and extension at 72?C for 1 min. PCR amplifications of 28S was performed using 1 ?l of primers A-D3A (5’-GCCTTGCCAGCCCGCTCAGGACCCGTCTTGAAACACGGA -3’) and B-D3B (5’-GCCTTGCCAGCCCGCTCAGTCGGAAGGAACCAGCTACTA-3’) and AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) using denaturation at 95?C for 5 min, followed by 35 cycles of denaturation at 95?C for 1 min, annealing at 55?C for 1 min, and extension at 72?C for 2 min. A final extension was identical for both PCR programs and was performed at 72?C for 10 min. Negative controls were included for each amplification. All PCR products along with 1Kbp DNA ladder (Promega, Madison, WI) were electrophoresed on 1% agarose gel (Fisher Biotech) and stained with 0.003% ethidium bromide. No PCR products were observed in the negative controls.  Positive bands of the correct size were assumed to be indicative of the presence of the nematode diagnostic amplicons, although the presence of fungal and other contaminants could not be excluded. PCR products were purified using Montage PCR Centrifugal Filter Devices (Millipore Corp., Bedford, MA) and visualized again on the agarose gel.  All 18S PCR reactions yielded positive bands, however, eight 28S bands were missing (Table 1). Five ?l of PCR products from all single nematode PCR reactions were pulled together to create two metagenomic samples: 18S sPCR and 28S sPCR. Two additional metagenomic samples consisted of DNA amplified via multiple nematode PCR reactions and were labeled: 18S mPCR and 28S mPCR.    Approximately 15 ?l of each sample was shipped on ice to the Interdisciplinary Center for Biotechnology Research (ICBR), 454 Sequencing for DNA quantification, calibration, and sequencing. All metagenomic samples were run separately on a 16th-chambered plate. All PCR products were A-Amplicon sequenced using 454 proprietary primers on GS FLX (454 Life Sciences, Roche Applied Science). Generated sequences were first screened against our database. In case of no matches, data were compared to published data in GenBank. A sequence data base of the 41 nematode species was constructed by amplifying each nematode species individually under conditions as described above and sequencing at the DNA Sequencing Core Laboratory of the University of Florida ICBR using dideoxy termination procedure on an automated DNA sequencer Model 377 (Applied Biosystems).