Lasergene genomics suite

Genomic DNA was prepared using the Qiagen DNeasy Blood and Tissue Kit and submitted for Mi-Seq analysis (Laragen). The data were analyzed using the DNASTAR Lasergene Genomics Suite (DNASTAR). FastQ files were assembled with the SeqMan NGen module using B. fragilis 638R as the template sequence (638R_NC016776.1; GI : 375356399) and the default parameters. Comparisons of the aligned files were visualized using the SeqMan Pro module and areas of conflict are shown. Detection of prophage sequences within the genome was done with the Phaster tool [34, 35 (link)].

The raw RNA-Seq reads (FASTQ) were processed and analyzed using Lasergene Genomics Suite (DNASTAR, Madison, WI, USA). The paired-end reads were assembled with SeqMan NGen (Ver. 12), using default parameters and aligned to the human reference genome (GRCh38.p11). The ArrayStar (Ver. 12; DNASTAR) was used for normalization, differential gene expression and statistical analysis of mapped paired-end reads using the default parameters. The expression data quantification and normalization were calculated using the RPKM value for each gene^{21 (link)}.
In parallel, we examined differential expression in hESC- and iPSC-derived lentoid bodies using Spotfire DecisionSite with Functional Genomics (TIBCO Spotfire, Boston, MA). Expression differential between hESC- and iPSC-derived lentoid bodies were analyzed to determine standard deviation from their mean of 0, which represents no change. We chose > 2 Std. Dev. the cutoff value for differentially expressed genes.

We assembled IBV genomes by aligning raw Illumina reads to GI-11 and G-16 Uruguayan strains (MF421320 and MF421319, respectively) using DNASTAR’s Lasergene Genomics Suite with the default workflow (DNASTAR, Madison, WI, USA). Assemblies were visually inspected and manually optimized to obtain a single contig. Genome sequences obtained were aligned with MAFFT [40 ], and annotations were transferred from reference strains and manually curated using Megalign Pro (DNASTAR).
The sequences were deposited in the GenBank database; the accession numbers are given in Table 1.

Fully sequenced and assembled Listeria phage genomes used for comparison, including the sequences of M188 and MC293, were obtained from the NCBI database (http://www.ncbi.nlm.nih.gov/genome). Linear genome comparisons of these phages were undertaken using Easyfig (Sullivan et al., 2011 (link)). For each input phage in this analysis, the protein products of each coding sequence were concatenated into a single file. Progressive multiple protein sequence alignments were then performed via the slow-accurate ClustalW method using the Gonnet series protein weight matrices, and were generated in the MegAlign application of the Lasergene Genomics Suite (DNAStar Inc., Madison, USA). A phylogenetic tree of the alignment was generated in MegAlign using the Kimura distance formula, with bootstrap analysis performed using default parameters of trial number = 1000 and random seed number = 111. HHpred analyses of protein sequences were undertaken using the online bioinformatics toolkit software on the HHpred interactive server (http://toolkit.tuebingen.mpg.de/hhpred).

Mid-log cells of B. fragilis grown on BHI broth were harvested and RNA was prepared using the RNeasy minikit with RNAprotect bacterial reagent (QIAGEN, Valencia, CA). Purified total RNA was again treated with RNase-free DNase kit (QIAGEN, Valencia, CA). Following RNase-free DNase treatment, reduction in genomic DNA in the RNA sample was confirmed by qRTPCR; RNase-free DNase treatment effectively reduced genomic DNA contamination by >1000 fold. The majority of the rRNA (>95%) was removed from total RNA using the MICROBExpress™ Bacterial mRNA Enrichment Kit (Life Technologies Corporation) leaving enriched RNA. The cDNA was prepared from enriched mRNA using the SuperScript® Double-Stranded cDNA Synthesis Kit (Invitrogen™) and subjected to RNA-Seq at Otogenetics (Norcross, USA). The RNA-Seq files were analyzed using the Lasergene Genomics Suite (DNASTAR, Inc, Madison, USA).