DNA isolation Milk samples (1 mL) were centrifuged at 5,000 × g for 10 minutes to pellet eukaryotic cells. Prokaryotic cells were pelleted from milk serum by centrifugation at 13,000 × g for 15 minutes. HSP990 pellets were resuspended in 2 mL phosphate buffered saline with 1% Triton X-100 and incubated for 2 hours at 37°C to lyse any selleck chemicals remaining eukaryotic cells. Bacteria were pelleted by centrifugation at 13,000 × g for 15 minutes and pellets were resuspended in 500 μL TE with 30 μL of 10% sodium dodecyl sulfate and 5 μg proteinase K. Samples
were incubated for 2 hours at 37°C, and DNA was isolated using phenol/chloroform as previously described . DNA pellets were resuspended in 50 μL TE buffer and pooled. A total of ~4 μg of double stranded DNA was isolated as quantified with Quant-iT PicoGreen (Invitrogen, Burlington, ON, Canada) using a Typhoon Trio Imager and Image Quant TL software (GE Healthcare, JQ-EZ-05 mw Waukesha, WI, USA). DNA integrity was also determined by agarose gel electrophoresis prior to sequencing. DNA sequencing, filtering and contig assembly The pooled DNA sample was sequenced seven independent times by StemCore Laboratories (Ottawa, Ontario, Canada). DNA was prepared according to the DNA sample preparation protocol 1003806 Rev. B for Illumina sequencing (Illumina Inc, San Diego, CA, USA). Sequencing was performed using an Illumina GAIIx Genome Analyzer and Illumina CASAVA analysis pipeline
(v 1.7.0). Sequences were aligned to the human genome (hg19/NCBI37) with a stringency of 2 bp mismatching using ELAND (Illumina Inc). Prokaryotic genomes (1,731 genomes) were imported from NCBI. Sequences were oxyclozanide aligned to the genomes using BLAT (Kent Informatics, Inc.) and sorted via best hit analysis to genera according to “List of Prokaryotic Names with Standing in Nomenclature” (http://www.bacterio.cict.fr/, accessed February 2012). Unidentified sequences were further filtered by using BLAT against the human genome with a stringency of ≤10 mismatches or gaps. Both prokaryotic and remaining unknown sequences were assembled into contigs using Ray v1.7 . Contigs, ORF prediction and characterization
Assembled contigs were uploaded to the MG-RAST pipeline . Organism abundance was analyzed using a lowest common ancestor approach with a maximum e-value of 1 × 10-5, a minimum identity of 60%, and a minimum alignment length of 15 measured in amino acids for protein and base pairs for RNA databases. A functional abundance analysis of ORFs was performed using “”Hierarchical Classification”" by comparing to subsystems with a maximum e-value of 1 × 10-5, a minimum identity of 60%, and a minimum alignment length of 15 measured in amino acids for protein and base pairs for RNA databases. Previously reported and publicly available metagenomes of feces from five unrelated BF-infants, five FF-infants (metagenome IDs: USinfTW4.1, 6.1, 10.1, 11.1, 12.1, 13.1, 15.1, 19.1, 20.1, and 21.