title: Whole genome sequencing of B. thetaiotaomicron 7330 using the PacBio RSII platform
design: High molecular weight genomic DNA was extracted from an overnight culture using a hexadecyltrimethylammonium bromide (CTAB)/chloroform-isoamyl alcohol genomic DNA extraction protocol and then sheared to 10-20 kb fragments using Covaris g-TUBES. Sequencing libraries were prepared with 5 μg of the sheared DNA according to the “20 kb Template Preparation with 15kb Cutoff Using BluePippin™ Size-Selection” protocol (https://pacbio.secure.force.com/Share/Protocol?id=a1q70000004GlScAAK) and run on the Pacific Biosciences RSII sequencer with 240 minutes movies and the P6/C4 chemistry. Two Single Molecule Real Time (SMRT) cells were used, yielded 119,018 reads and 1,214 MB of sequence data. Reads were assembled using PacBio’s hierarchical genome-assembly process (HGAP) 2.0. Polymerase reads with quality score lower than 0.80 were filtered and remaining high quality reads were processed into subreads by removing adaptor sequences. Subreads longer than 500 bp were retained as input for the HGAP assembler with subreads longer than 4kb treated as seeds. The error-corrected pre-assembled reads were used with the Celera Assembler to produce two large contigs of 5.0 MB and 1.5 MB. Two inverted repeats of 1,204 bp and 10,160 bp were found during finishing at the ends of the two large contigs. Pre-assembled long reads provided evidence that both orientations of the unique sequence between the two inverted arms were present in the bacterial culture; the orientation with the most reads was selected for the final closed circular reference assembly of 6,487,685 bp after Illumina indel error correction.