Longitudinal study of murine gut microbiota activity and interactions with the host during acute inflammation and recovery-14
"Double stranded cDNA libraries were paired-end sequenced using an Illumina HiSeq (Campus Science Support Facilities GmbH, Vienna). Read pairs were overlapped using FLASH, which yielded reads of approx. 170 bp (Mago? and Salzberg, 2011). Metatranscriptomic sequencing data were analyzed following an established double RNA analysis pipeline (Urich et al., 2008). Approximately 95% of the reads per sample were derived from rRNA; the number of putative mRNA reads per sample ranged from 75,305 to 1,166,930 . Community composition was determined from 100,000 randomly chosen rRNA reads per sample, which were taxonomically assigned using CREST (Lanz?n et al., 2012) (bit score=150, top percent=10, minimal support=5). mRNA tags were compared against the NCBI RefSeq database using BlastX and functionally and taxonomically classified using MEGAN and the SEED functional classification scheme therein (bit score=40, top percent=10, minimal support=1) (Mitra et al., 2011). This resulted in 24,190 to 368,950 functionally annotated mRNAs. Metatranscriptomes were generated from three replicates collected on days 1, 5, and 14 and from four replicates collected on days 8 and 25."