Amplicon sequencing utilizing next-generation platforms has significantly transformed how research is conducted, specifically microbial ecology. However, primer and sequencing platform biases can confound or change the way scientists interpret these data. The Pacific Biosciences RSII instrument may also preferentially load smaller fragments, which may also be a function of PCR product exhaustion during sequencing. To further examine theses biases, data is provided from 16S rRNA rumen community analyses. Specifically, data from the relative phylum-level abundances for the ruminal bacterial community are provided to determine between-sample variability. Direct sequencing of metagenomic DNA was conducted to circumvent primer-associated biases in 16S rRNA reads and rarefaction curves were generated to demonstrate adequate coverage of each amplicon. PCR products were also subjected to reduced amplification and pooling to reduce the likelihood of PCR product exhaustion during sequencing on the Pacific Biosciences platform. The taxonomic profiles for the relative phylum-level and genus-level abundance of rumen microbiota as a function of PCR pooling for sequencing on the Pacific Biosciences RSII platform were provided. Data is within this article and raw ruminal MiSeq sequence data is available from the NCBI Sequence Read Archive (SRA Accession SRP047292). Additional descriptive information is associated with NCBI BioProject PRJNA261425. http://www.ncbi.nlm.nih.gov/bioproject/PRJNA261425/ Resources in this dataset:Resource Title: NCBI Sequence Read Archive (SRA Accession SRP047292). File Name: Web Page, url: https://www.ncbi.nlm.nih.gov/sra/SRX704260 1 ILLUMINA (Illumina MiSeq) run: 978,195 spots, 532.9M bases, 311.6Mb downloads.