There, a 410-420 bp fragment spanning two variable regions (V4 and V5) in 16s rDNA genes was amplified using the primers 519F 5′-CAGCAGCCGCGGTAATAC-3 and 926R 5′-CCGTCAATTCCTTTGAGTTT-3, targeting Bacteria. To increase the number of reads, all samples were run as multiplex on the same ¼ picoplate using nucleotide barcodes tags on primers, allowing sample identification to each sequence read. Analysis of data
from pyrosequencing All sequences in the output file from the FLX sequencer was sorted into sample groups based on the barcode tag. After trimming all sequences for barcodes and fusion primers using the FLS software, sequences were imported into the CLC bio software (CLC bio, Aarhus, Denmark), where they were checked, aligned and filtered for high Abemaciclib order quality sequences. OTU’s were generated by CLC TSA HDAC molecular weight based on 99% similarity on the data set that had a sequence longer than 400 bp. The Sequence match analysis tool in the Ribosomal database project 10 http://rdp.cme.msu.edu/ was used to assign the phylogenetic position of each OTU. The search criteria were for both GNS-1480 type and non-type strains, both environmental (uncultured) sequences and isolates, near-full-length sequences (> 1200 bases) of good quality. If there was a consensus at the genus level, the
tag was assigned this taxonomic classification. If no such consensus was found, the classification proceeded up one level to family, and again
if no taxonomic affiliation could be assigned the tag continued to be proceeded up the tree, as described by Huse et al. [32]. In some cases, it was not possible to assign a domain, and these sequences might represent new organisms or the sequences might be biased; in these cases the tags were excluded from the dataset. In total 250,007 sequences were finally assigned a taxonomic classification in this study. Acknowledgements This work was founded under the European Union Framework Program 6, under contract 065547 (Safehouse Project). We would like to thank Annie Brandstrup and Lis Nielsen for excellent technical assistance. References 1. Tauson R: Management and GBA3 housing systems for layers – effects on welfare and production. World Poultry Sci J 2005, 61:477–490.CrossRef 2. Tauson R: Furnished cages and aviaries: production and health. World Poultry Sci J 2002, 58:49–63.CrossRef 3. De Reu K, Grijspeerdt K, Heyndrickx M, Zoons J, De Baere K, Uyttendaele M, Debevere J, Herman L: Bacterial eggshell contamination in conventional cages, furnished cages and aviary housing systems for laying hens. Brit Poultry Sci 2005, 46:149–155.CrossRef 4. Corrier DE, Nisbet DJ, Hargis BM, Holt PS, DeLoach JR: Provision of Lactose to Molting Hens Enhances Resistance to Salmonella enteritidis Colonization. J Food Protec 1997, 60:10–15. 5.