1. Convenience Sample of 4 Adults and 6 Infants. Adults 4 visits over 2 weeks; infants 2 visits over 2 weeks Adult specimens: 1) plaque (by method, teeth, and media) 2) saliva (by media & storage temperature) Infant Specimens: 1) plaque (by storage temperature & number of teeth) 2) saliva (by storage temperature) 3) gum (by storage temperature) Adult plaque have biological replicates Adult saliva are technical replicates All Infant specimens stored in Omnigene MethodsConclusions Contact & Support Email tingluo@umich.edu Supported by NIDCR R01-DE014889 PIs: Marazita [U PITT]; McNeil [West Virginia U]; Foxman [U of Michigan] Validation of Specimen Collection Methods for 16S Oral Microbial Community Analysis Ting Luo 1, Usha Srinivasan 1, Kirtana Ramadugu 1, Kerby Shedden 1, Erika Trumble 1, Jiean Li 1, Katherine Neiswanger 2, Richard Crout 2, Daniel McNeil 2, Robert Weyant 3, Mary L. Marazita 3, Betsy Foxman 1 1 University of Michigan, 2 West Virginia University, 3 University of Pittsburgh Poster Presented at the International Association for Dental Research conference Boston, MA, USA; March, 2015 4083 Introduction and Research Questions The effects of different specimen collection, media and storage conditions on the recovery of bacterial DNA for exploring the oral microbiome have not been examined in detail. 16S metagenomics requires recovery of high quality sequences in large abundances for accurate characterization of the oral microbiome. Our objective was to validate various specimen collection methods for adequate bacterial DNA recovery for Illumina sequencing and 16S metagenomic analyses. Results Introduction and Research Questions Study Population & Sample Collection Design Barcoded PCR Fastq files Preprocessing (remove barcodes, Primers and quality scores) Pick Open Reference OTUs (Uclust) Reference based clustering w/ CORE dataset (RDP Classifier) Sequence Alignment (PyNast) Beta Diversity & Normalized Counts (Weighted UniFrac, Euclidean & Nonmetric Distances) Variance Component Model (Linear mixed effects regression) Import Prune/Subset Data Inference Validation Graphics Library generation Sample DNA Extraction (Qiacube®) Illumina Sequencing QIIME Upstream Pipeline Alpha Diversity & Boostrapping (sequencing depth, Observed OTUs) Sample preparation, Sequencing and analysis pipeline Barcode to Sample Sample to Metadata Primers to Sample Relative Abundance & Unrarefied Data (Phyla Level, Wald Negative Binomial Testing) 1)16S rRNA amplified using 8 base pair error-correcting barcodes with conserved primers flanking the V6 region 2)Amplicons from each sample pooled and sequenced with Illumina HiSeq Platform (100-cycle paired end reads) 208,364,761 raw mate pairs generated 202,312,951 mate pairs successfully joined by FLASH 171,758,509 joined reads demultiplexed and passed quality filtering # of reads mapped to a sample ranged from 240 – 9,533,688 147/152 samples have over 10,000 reads 3) Processed reads fed into QIIME pick open reference OTU workflow 217,308 OTUs picked, 749 above.005% relative abundance 4) Rare OTUs (<.005% relative abundance) filtered and resultant OTU table imported into Phyloseq with metadata OTU Table Taxonomy Table Phylogenetic Tree Reference Sequences Phyloseq Upstream Pipeline Adult PlaqueAdult Saliva VisitMediaMethodVisitMediaMethodStorage 1LDTMScaler (17, 26) molars1OmnigeneSubject Spitting-20C 1LDTMScaler (34, 45) premolars1OmnigeneSubject SpittingRT for 2 days 1OmnigeneScaler (36, 47) molars1OmnigeneSubject SpittingRT for 5 days 1OmnigeneScaler (15, 24) premolars1OmnigeneSubject SpittingRT for 7 days 1LDTMBrush (16, 27) molars1LDTMSubject Spitting-20C 1LDTMBrush (44, 35) premolars1LDTMSubject SpittingRT for 2 days 1OmnigeneBrush (46, 37) molars1LDTMSubject Spitting-80C for 2 days 1OmnigeneBrush (14, 25) premolarsInfant Saliva 2LDTMScaler (17, 26) molars1OmnigeneSwab-20C 2LDTMScaler (34, 45) premolars2OmnigeneSwabRT 2OmnigeneScaler (36, 47) molarsAdult Gum 2OmnigeneScaler (15, 24) premolars1OmnigeneSwab-20C 2LDTMBrush (16, 27) molars2OmnigeneSwabRT 2LDTMBrush (44, 35) premolarsInfant Plaque 2OmnigeneBrush (46, 37) molars1OmnigeneBrush (1 tooth)-20C 2OmnigeneBrush (14, 25) premolars2OmnigeneBrush (1 tooth)RT 3LDTMScaler (36, 37, 26, 27)1OmnigeneBrush (4 teeth)-20C 3LDTMBrush (17, 16, 46, 47)2OmnigeneBrush (4 teeth)RT 4OmnigeneScaler (36, 37, 26, 27) 4OmnigeneBrush (17, 16, 46, 47) Scaler vs. Cytobrush Omnigene vs. LDTM 3) Do different storage temperatures affect bacterial DNA recovery from saliva? 4) Are bacterial communities in plaque reflected in the saliva for adults? 5) Are bacterial communities in plaque reflected in the gum and/or saliva for infants? Omnigene recovered more taxa than LDTM Bacterial communities are similar for scalar and cytobrush Omnigene had advantages over LDTM in terms of convenience and sensitivity to storage conditions Recovery was similar by scaler or cytobrush The number of taxa recovered varies by specimen Adult Plaque Actinobacteria Bacteroidetes Firmicutes Fusobacteria OtherProteobacteria Spirochaetes TM7 Random EffectsVariance Replicate: Participant9.00.0 0.319.20.0010.000 Replicate9.26.83.50.51.50.00.0000.004 Residual131.583.344.21.47.486.60.0010.022 Fixed Effects Estimate (t-value) Estimate Intercept12.7(3.9)7.8(3.1)38.5(21.0)1.4(3.1)2.2(2.4)37.2(14.1)0.052(3.117)0.151(3.159) Omnigene7.3(2.7)9.0(4.4)-7.7(-5.2)0.3(1.1)0.1(0.1)-9.2(-3.8)0.024(1.865)0.093(2.816) Scaler-2.8(-1.0)0.3(0.1)1.0(0.7)-0.3(-1.0)1.6(2.5)0.3(0.1)-0.008(-0.620)-.022(-.658) Pooled Plaque-0.9(-.3)2.7(1.0)1.5(.8)0.5(1.5)-1.0(-1.2)-2.8(-0.9)-0.017(-1.030)0.010(0.223) Premolar Plaque3.8(1.3)-1.4(-0.6)-1.6(-1.0)0.1(0.2)0.2(0.3)-0.9(-0.3)-0.030(-1.973)-.065(-1.771) Infant Samples Random EffectsVariance Participant6.60.0 0.30.20.00.0000.001 Residual59.362.3107.82.04.8128.10.0010.013 Fixed Effects Estimate (t-value) Estimate Intercept10.5(3.7)25.1(9.6)30.2(8.8)2.0(4.0)2.8(3.7)29.3(7.8)0.044(4.175)0.172(4.424) Plaque From 1 Tooth1.7(0.5)-7.2(-2.2)4.5(1.0)-0.3(-0.5)0.8(0.9)0.5(0.1)-0.002(-0.116)-.001(-.028) Plaque From 4 Teeth5.0(1.5)-7.8(-2.3)-3.4(-0.8)-0.1(-0.1)-1.5(-1.6)7.8(1.6)0.003(0.214)0.024(0.485) Infant Saliva1.0(0.3)-8.4(-2.5)5.4(1.2)-0.4(-0.7)-1.9(-2.1)4.4(0.9)-0.033(-2.461)-.104(-2.182) Storage at -20C -2.1(-0.9)-6.9(-2.8)6.0(1.9)-0.1(-0.2)-0.9(-1.3)4.0(1.2)-0.010(-1.048)-.072(-2.097) Bi-variate results confirmed after taking into account random and fixed effects 1) Do dental scalers recover different bacterial communities from plaque than cytobrushes? 2) Do the bacterial communities recovered differ by storage media? Bacterial communities recovered differ among plaque, saliva and gum, and media Omnigene was less sensitive to storage conditions Number of taxa recovered depended on specimen sampled