Nebnext dna library prep master mix set for 454

Each faeces pellet was dissolved in 500 μl of PBS. Two different kits were employed for DNA extraction from the dissolved pellets, the QIAamp DNA Stool for Pathogen detection and the QIAamp DNA Mini and Blood (Qiagen), according to the manufacturers' instructions. The DNA was subsequently passed through a MinElute spin column (Qiagen) and eluted in 20 μl EB buffer. DNA concentration was measured on the Qubit® 2.0 Fluorometer (Invitrogen).
The DNA extracts from each sample where digested with 15 U of Plasmid-Safe™ ATP-dependent DNase (Epicentre) in the presence of 112.5 nmol ATP. The remaining DNA was amplified using the REPLI-g® Midi kit (Qiagen) according to the manufacturers' instructions. A total of 2 μg of DNA was fragmented on the Bioruptor® NGS (Diagenode) to an average length of 300 bp, as verified by running the samples on a High Sensitivity chip on the Agilent 2100 Bioanalyzer instrument. Libraries were built with the NEBnext® DNA Library Prep Master Mix Set for 454 (New England Biolabs) kit, with some modifications. Paired-end sequencing was performed on the Illumina HiSeq 2000 instrument.

BAC clones were sequenced using Roche GS FLX (Basel, Switzerland). BAC DNAs were prepared in a 96-deepwell plate, extracted using the alkaline lysis method and purified with the MultiScreen Filter plate (Millipore, Billerica, MA, USA). After fragmentation with the Covaris Acoustic Solubilizer LE220 (Covaris, Woburn, MA, USA), the sequencing libraries were constructed using the NEBNext DNA Library Prep Master Mix Set for 454 (New England Biolabs, Ipswich, MA, USA) and tagged with Multiplex Identifier (MID) Adaptors (Roche, Basel, Switzerland). The tagged DNAs were subjected to size selection using agarose gel electrophoresis to obtain DNA fragments of the appropriate size (600–1200 bp) and then quantified using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). The resulting libraries were sequenced according to the manufacturer’s recommendations. The resulting SFF files were split into separate files of each BAC clone based on MID by using the sffile program in SFF Tools (Roche, Basel, Switzerland). The split SFF files were used for de novo assembly with the GS De Novo Assembler v2.6 (Roche, Basel, Switzerland) with default parameters, and the BAC vector and E. coli genome sequences were trimmed.