Real time analysis software

Base call (.bcl) files for each cycle of sequencing were generated by Illumina Real Time Analysis (RTA) software. The base call files and run folders were then exported to servers maintained at the Minnesota Supercomputing Institute. Primary analysis and de-multiplexing were performed using Illumina’s CASAVA software 1.8.2. The end result of the CASAVA workflow was de-multiplexed FASTQ files.

Libraries were prepared from poly-adenylated mRNA enriched from total RNA samples (200ng-1ug with RIN> 8) using the Illumina Truseq RNA prep kit. Libraries were sequenced in multiplex targeting 30 million single-end 100bp reads per sample on an Illumina HiSeq2000 at the Columbia University Genome Center. The Illumina Real Time Analysis (RTA) software was used for base calling followed by bcl2fastq (v.1.8.4) for adaptor trimming. Tophat (v2.1.0) was used to map reads to the mouse UCSC/mm9 reference genome (<4 mismatches <10 multiple hits). HTSeq (v0.6.1) with default settings was used count reads against the UCSC RefSeq mm9 annotation file. Genes with expression of at least 1 count per million reads in at least one sample were then analyzed for relative abundance and differential expression using R(v3.2)/Bioconductor with package EdgeR (3.10.5). Multigroup gene comparisons were performed using a generalized linear model likelihood ratio test in EdgeR to find differential gene expression. A multidimensional scaling plot was created using expression data for those genes with at least 120 counts per million in at least 4 samples. The top 423 genes different (FDR < 0.05) between OIO and ALO were analyzed using Enrichr, a comprehensive gene set enrichment analysis web server. GO biological process lists were generated and the top ten were presented in table 2.

Metagenomic shotgun sequencing on prepared microbiota samples was performed at the University of Minnesota Genomics Center (UMGC). DNA samples were quantified using a fluorimetric PicoGreen assay gDNA samples were converted to Illumina sequencing libraries using Illumina’s NexteraXT DNA Sample Preparation Kit (Cat. # FC-130-1005). 1 ng of gDNA was simultaneously fragmented and tagged with a unique adaptor sequence. This “tagmentation” step is mediated by a transposase. The tagmented DNA was simultaneously indexed and amplified with 12 PCR cycles. Final library size distribution was validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen). Truseq libraries were hybridized to a NextSeq. Base call (.bcl) files for each cycle of sequencing were generated by Illumina Real Time Analysis (RTA) software. The base call files were demultiplexed and then converted to index specific fastq files using the MiSeq Reporter software on-instrument.