Login to Access Video or Poster Abstract: MP12-05
Sources of Funding: none

Introduction

High-throughput metagenomic profiling is becoming increasingly important in the field of urology. Analyses of urinary and gut microbiomes have been performed in the study of nephrolithiasis, prostate and bladder cancer, prostatitis and urinary incontinence. Various sequencing platforms exist in the study of the microbiome and may exhibit inherent biases. The selection of DNA sequencing platform can shape our understanding of taxonomic diversity in the study of urologic microbiomes and specifically in our understanding of nephrolithiasis. We compared the output of two high-throughput sequencing platforms in the analysis of a highly efficient oxalate-degrading microbiome.

Methods

Four Neotoma albigula, white-throated woodrats, were fed high and low oxalate diets ranging from 0.2-12% oxalate. Fecal samples were collected from each animal. The samples were frozen at -80°C until DNA extraction. MiSeq microbial inventories were generated by amplifying and sequencing the hypervariable V4 region of the 16S rRNA gene with primers 515F and 806R. HiSeq inventories were generated by extracting 16S rRNA sequences from shotgun metagenomic data of the same DNA samples with HMMER. Sequencing was conducted at the same laboratory (Argonne National Laboratory, Chicago, IL). After consolidating the data from both platforms, a de novo picking strategy was used to classify the operational taxonomic units (OTU). Alpha and beta diversity metrics were compared across platforms and time using open source software, QIIME and R. Significance was defined at a P value of <0.05.

Results

There were only 10 Oxalobacteraceae OTUs identified with the MiSeq platform compared to 128 identified with HiSeq. The alpha diversity metrics were significantly different across the MiSeq and HiSeq platforms. However these metrics were different across time only for MiSeq and not HiSeq. Beta diversity metrics demonstrated a significant difference across platforms but not across time for either MiSeq or HiSeq.

Conclusions

Our results indicate that differences between the Illumina MiSeq and HiSeq platforms are primarily the result of MiSeq under sampling of rare taxa. The MiSeq platform underestimated the diversity of the oxalate-degrading Oxalobacteraceae and exhibited significant compositional differences with the data derived from the HiSeq platform. The limitations of the MiSeq platform must be considered in microbiome studies of urologic disease and has implications for our understanding of the oxalate metabolism in nephrolithiasis.

Funding

none