16S rRNA sequence processing.

We performed 16S sequencing on fecal samples from Ldlr^−/− and ApoE^−/− mice for all time points. DNA extraction and 16S rRNA amplicon sequencing were done using Earth Microbiome Project (EMP) standard protocols (http://www.earthmicrobiome.org/protocols-and-standards/16s) (49). In brief, DNA was extracted using the Mo Bio PowerSoil DNA extraction kit (Carlsbad, CA). Amplicon PCR was performed on the V4 region of the 16S rRNA gene (Platinum Hot Start PCR 2× master mix; Invitrogen RED 13000014) using the primer pair 515f to 806r with Golay error-correcting barcodes on the reverse primer. Amplicons were barcoded and pooled in equal concentrations for sequencing. The amplicon pool was purified with the Mo Bio UltraClean PCR cleanup kit and sequenced on the Illumina HiSeq 2500 sequencing platform. Sequence data were demultiplexed and minimally quality filtered using the QIIME 1.9.1 script split_libraries_fastq.py, with a Phred quality threshold of 3 and default parameters to generate per-study FASTA sequence files.

The raw sequence data were processed using the Deblur workflow (23) with default parameters in Qiita (50). This generated a sub-operational taxonomic unit (sOTU) abundance per sample (BIOM format) (51). Taxonomies for sOTUs were assigned using the sklearn-based taxonomy classifier trained on the Greengenes 13_8 99% OTUs (feature classifier plug-in) in QIIME 2 (52). The sOTU table was rarefied to a depth of 2,000 sequences/sample to control for sequencing effort (53). A phylogeny was inferred using SATé-enabled phylogenetic placement (54), which was used to insert 16S Deblur sOTUs into Greengenes 13_8 at a 99% phylogeny.