454 genome sequencer flx platform

Total RNA was extracted from cheese surface smear samples applying the protocol described by Monnet and colleagues [45] and converted to cDNA. Subsequently the cDNA was sheared and the region corresponding to the 16S rRNA was selectively enriched by a set-up called vectorette [46] (link), using the universal primer U515F as specific primer and 13 cycles of amplification. After size selection (about 600 bp) the libraries were sequenced using GS FLX Titanium chemistry on a 454 Genome Sequencer FLX platform (Roche Diagnostics Ltd., Burgess Hill, West Sussex, United Kingdom) according to Roche 454 protocols. Sequences not passing the FLX quality controls were not considered for bioinformatics analysis, the 454-specific portions of the primers were trimmed, the raw sequences were sorted according to tag sequences and reads with low quality scores (below 40) and short lengths (less than 200 bp) were removed. The resulting 16S rRNA reads were used for subsequent taxonomic classification of the metabolic active cheese surface microbiota as described previously for the sequencing of the 16S rDNA amplicons.

Approximately 1 μg total RNA was used for preparing mRNA sequencing library of each sample. Poly (A⁺) RNA was isolated from total RNA mixture by using NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), following the manufacturer’s protocol. The purified Poly (A⁺) RNA was sheared and cDNA library was prepared by using NEBNext® mRNA Library Prep Reagent Set for 454™ (New England Biolabs) as per the instruction manual. The cDNA libraries were sequenced on a 454 Genome Sequencer FLX+ platform (Roche, USA).

Genomic DNA was extracted from ~ 0.3 g of each sample in duplicate using the FastDNA SPIN Kit for Soil (Qbiogene-MP Biomedicals, Irvine, CA, USA), according to the manufacturer's instructions. The V3-V5 hypervariable region of 16S rDNA genes was amplified using the primers 907F (5′-CCGTCAATTCMTTTGAGTTT-3′) and 338R (3′–ACTCCTACGGGAGGCAGCAG-5′). Each forward primer with a unique 10 bp barcode was used to tag each sample. The amplification mixture contained 25 μL Failsafe Premix F (Epicentre Biotechnologies, Madison, WI, U.S.A.), 0.4 μM each primer, 2.5 U of Ex Taq DNA polymerase (Takara, Dalian, China) and 1–2 μL DNA template in a total volume of 50 μL. PCR reactions were performed at 95°C for 5 min, followed by 25 cycles of 95°C for 30 s, 56°C for 30 s and 72°C for 90 s, and final extension at 72°C for 7 min. All samples were amplified in triplicate, pooled, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). Pyrosequencing was performed on a 454 Genome Sequencer FLX platform (Roche) using the GS FLX Titanium XLR70 sequencing kit according to Roche Sequencing Method (Manual GS FLX Titanium Series October 2009 Edition). All DNA extraction, amplification and pyrosequencing procedures were operated by a professional at BGI (Shenzhen, China).