Metagenome from lower respiratory tract of birds
Preparation of PCR amplicons (dsDNA) and 454-sequencing One-way read amplicons (Lib-L) were prepared using bar-coded fusion primers withthe 27F (5´AGAGTTTGATCMTGGCTCAG 3´) and 907R (5´TACGGGAGGCAGCAG 3´) 16S rRNAprimers (forward: CCATCTCATCCCTGCGTGTCTCCGACTCAG-MID-AGTTTGATCMTGGCTCAG and reverse: CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-TACGGGAGGCAGCAG). The PCR was performed onV1–V5 region of 16S rRNA. The samples were denatured at 94°C for 3 minutes, followed by 35 cycles of 94°C for 15 sec, 55°C for 45 seconds, and 72°C for 60 seconds and a final extension at 72°C for 8 min (Gene AMP PCR System 9700; Applied Biosystems, Foster City, CA). The PCR products (approximately 900 bp) were separated on agarose gels and were extracted and purified using AgencourtAMPure technology (Beckman Coulter, Brea, CA). This technique is important for the removal of undesirable shorter-length DNA fragments and improved sequencing yield. After purification, the products were quantified using both Qubitflourometer (Lifetech, Carlsbad, CA) and qPCR using the KAPA Biosystems library quantification kit (KapaBiosystems, Woburn, MA). The products were pooled based on the molar amounts, separated on a 1% agarose gel and extracted. After purification, using the QIAquick PCR purification kit (Qiagen,Valencia, CA), the quality and quantity of the samples were assessed again using a DNA 7500 LabChip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and Qubitflourometerquantification.Pyrosequencing on a 454/Roche GS FLX+ instrument using titanium chemistry (Roche Diagnostics, Indianapolis, IN) was performed in accordance with the manufacturer’s instructions. Sequence processing The GS-FLX-Titanium sequencer data was generated (.sff file) with the GS Amplicon software package (Roche, Branford, CT). Collectively, 135,136 raw sequences with an average of 9,652 per sample were processed.Briefly, the primer sequences and barcodes were removed from each raw sequence, and the sequences were screened based on the quality score using the MOTHUR software platform v. 1.20.0. (37,38). Reads containing homo-polymers longer than 8 bp in a row, having any ambiguous bases, having one or more bp difference to the barcode sequence, or exhibiting an average quality score less than 25 were trimmed, and the individual bird sequence data were split using the split.groups command.After filtering, only those read longer than 100 bp were processed for subsequent analyses. Chimera sequences were identified and removed with chimera.slayer against mothur-adopted greengenes gold reference alignment. 