P-237 Multiple Double-barcoding 16S Sequencing on the MiSeq Platform to Study the Gut Microbiome in Ashkenazi Jews with Crohn’s Disease

BACKGROUND: Crohn’s disease (CD) results from defects in the mucosal immune response to luminal factors in genetically susceptible individuals. The role of the gut microbiome in CD pathogenesis has been suggested by several studies. Until recently, 454-pyrosequencing approach was considered the gold standard for microbiome studies. However, newer, more efficient, and cost-effective technologies are now available(1). Compared to other next-generation platforms, Illumina MiSeq has the advantage of higher throughput, better sequencing accuracy, and a shorter running time (24hrs). In this study, we designed a cost-efficient double-barcoding 16S rRNA sequencing using the MiSeq 2x250 pair-end method to evaluate the performance of MiSeq on 16S rRNA sequencing of the fecal microbiome of Ashkenazi Jews (AJ, a genetically homogeneous high-risk population) with and without Crohn’s disease. METHODS: Stool from 27 AJ-CD patients (in remission) and 16 AJ healthy controls was analyzed. We established a protocol for a multiple double-barcoding 16S rRNA sequencing using the MiSeq system (Fig. 1) and performed a taxon-based and phylogenetic analysis. Total DNA was extracted from the fecal samples and PCR-amplified with unique 8-bp barcoded primer sets targeting the 347-803 V3-to-V4 hypervariable regions. The 460bp PCR amplicons were pooled in equal molar amounts and sent for library preparation and sequencing. RESULTS: A single MiSeq run generated a total of ∼10 million paired reads. Our quality report revealed more than 95% reads with the average sequencing quality score passing Q30 and more than 50% reads with the quality of any individual base calling passing Q30. After merging, filtering by quality of the individual base calling and read length (>400bp), we obtained, on average, ∼10 thousand reads per sample. QIIME pipeline(2) was applied for taxonomy assignment (Fig. 2a) and diversity analysis. Repeated measurements of 8 subjects showed high correlations (r2 > 0.99). We observed a reduced diversity in the gut microbiota of CD patients (Fig. 2b). The NMDS and PerMANOVA test (Fig. 2c) using unweighted UniFrac distance matrix suggested that the overall microbiome of CD patients was significantly distinguished from healthy controls (p-value=0.002). We further compared the microbiome profile at various taxonomic ranks using the linear discriminant analysis (LDA) effect size (LEfSe) method (3). Compared to healthy controls, higher prevalence of Proteobacteria phyla was observed in CD patients (Fig. 3). At the genus level, Escherichia-Shigella, Parasutterella and Blautia were more abundant in the gut microbiota of CD patients, whereas the Parabacteroides and Oscillibacter were less abundant (Fig. 3). CONCLUSIONS: Using a cost-efficient multiple double-barcoding 16S rRNA sequencing method on the MiSeq platform, we herein report differences in microbial diversity and composition between CD and controls. These findings are consistent with previous studies (4), suggesting that our method is promising to conduct future microbiome studies. Supported by a CCFA Career Development Award (JH) and grants from The Chemotherapy Foundation (SI), New York Crohn’s Foundation (IP), and the NCI UH3CA140233, R01CA159036, and R03CA159414 (ZP), and Department of Veterans Affairs New York Harbor Healthcare System (ZP). References: [1] Nat Rev Genet. 2011;13:47-58 [2] Nature Methods. 2010;7:335-336 [3] Genome Biology 2011;12:R60 [4] Genome Biology 2012;13:R79. Figure. No caption available. Figure. No caption available. Figure. No caption available.