Diet & phylogeny shape Antarctic seal gut microbiota

Faecal samples were collected from southern elephant seals (n=24) and leopard seals (n=12) in the western Antarctic using rectal swabs. Sterile swabs (Oxoid Australia Pty, SA, Australia)were used and samples were frozen after collection. Faecal samples were collected opportunistically after defecation from captive leopard seals (n=2) housed at Taronga Zoo, Sydney, NSW, Australia. Total genomic DNA was extracted using QIAamp Stool DNA Mini Kit (Qiagen Pty, Vic., Australia) following the manufacturers protocol. Pyrosequencing of the16S rRNA gene was carried out at the Research and Testing Laboratory (Lubbock, TX, USA). Primers 27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 519R 5′-GTATTACCGCGGCTGCTG-3′ were used (Lane et al., 1985, PNAS USA, 82(20):6955-6959). PCR reactions were carried out individually for each sample using ~ 10 ng of template DNA. Tagged primer pairs and PCR reaction conditions followed the protocol of Dowd et al., 2008 (Foodborne Pathogens & Disease, 5(4):459-472). Sequencing was carried out on a Roche 454 titanium sequencer. Sequence data were trimmed to between 200 and 600 bp and labelled using the Mothur v.1.13.0 suite of programs (Schloss et al., 2009, Applied & Environmental Microbiology, 75(23):7357-7541).