Bioinformatics – Sequence Processing

The resulting paired-ended V4–V5 16S rRNA gene reads were assembled into contigs with the python script multiple_join_paired_ends.py by using QIIME software (version 1.9.0) (Caporaso et al., 2010). Then the contigs were curated using the QIIME script split_libraries.py with default parameters in order to assign contigs to samples and to remove low-quality (Q19 was the minimum acceptable quality score) contigs. UCHIME algorithm (Edgar et al., 2011) was used to remove chimeric sequences generated during the process of DNA amplification. The totality of filtered contigs were clustered into operational taxonomic units (OTUs) with a 97% similarity threshold using the QIIME script pick_open_reference_otus.py with default parameters (Rideout et al., 2014) that grouped, through UCLUST algorithm (Edgar, 2010), sequences against Greengenes reference database (version gg_13_5_otus) and also made a de novo clustering of those that did not match the database. The generated OTU table was filtered at: (1) sample level by discarding samples with less than 5,000 final contigs and at (2) OTU level by removing OTUs with less than 0.01% counts across samples. Finally, OTU table was normalized using the Cumulative Sum Scaling (CSS) method proposed by Paulson et al. (2013) yielding the normalized abundances of 596 OTUs for 43 samples. Note that three samples (cecal and fecal collected from one rabbit of size class “big” fed under restriction and cecal from another rabbit also of size class “big” and fed under restriction) did not pass the established threshold defined during the edition and quality control processes. In addition to this, in order to always keep parity between samples, i.e., for each animal to have both cecal and fecal samples, one fecal sample (from a rabbit of size class “big” fed under restriction) passing quality control was finally discarded for the next statistical analyses. Therefore, final analyses comprised of both types of samples (hard feces and cecum) from 21 animals. Taxonomic assignment of representative sequences of each OTU defined (596) was conducted by mapping them to the Greengenes reference database gg_13_5_otus with the UCLUST consensus taxonomy assigner (QIIME default parameters). The raw sequence data were deposited in the sequence read archive of NCBI under accession number (SRP149070).