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Improved Accuracy from Full-Length 16S Multiplexed Strain Identification Mock Community Classification

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Improved Accuracy from Full-Length 16S Multiplexed Strain Identification Mock Community Classification Accurately Surveying Uncultured Microbial Species with SMRT® Sequencing B. Bowman1, J-F. Cheng2, E. Singer2, D. Coleman-Derr2, S. Tringe2, S. Hallam3, T. Woyke2 1Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, 2Dept. of Energy Joint Genome Inst., Walnut Creek, CA, 3Univ. of British Columbia, Vancouver, BC, Canada Introduction Improved Accuracy from Full-Length 16S Multiplexed Strain Identification Mock Community Classification Analysis of bacterial Mock Pilot project for multiplexed Species OTU Count Concordance V4 Full Microbial ecology is reshaping our understanding of the natural world by revealing the large w/ Reference Class. Class. Community, composed of a strain identification from phylogenetic and functional diversity of microbial life. However the vast majority of these A Clostridium perfringens 1 99.92% Genus Species mixture of 22 bacterial species barcoded full-length 16S microorganisms remain poorly understood, as most cultivated representatives belong to just four Clostridium thermocellum 1 99.86% Genus Strain for which reference assemblies sequence data using phylogenetic groups and more than half of all identified phyla remain uncultivated. Characterization Coraliomargarita was available. Full-length 16S asymmetric, paired barcodes akajimensis 1 100% Genus Strain of this microbial ‘dark matter’ will thus greatly benefit from new metagenomic methods for in situ amplicons were generated with analysis. For example, sensitive high throughput methods for the characterization of community and Long Amplicon Analysis. Corynebacterium glutamicum 1 99.79% Genus Strain the 27F/1492R primer pair and composition and structure from the sequencing of conserved marker genes. Desulfosporosinus sequenced on the PacBio RS II. Figure 6 (Left-Top): acidiphilus 1 100% Genus Strain Sequence data was analyzed Here we utilize Single Molecule Real-Time (SMRT®) sequencing of full-length 16S rRNA amplicons Histogram of post-filter Desulfosporosinus meridiei 1 99.67% Genus Strain with rDnaTools[3]. B Desulfotomaculum to phylogenetically profile microbial communities to below the genus-level. We test this method with subread counts by barcode. gibsoniae 5 98.2%-100% Family Strain three different data sets: a mock community of known composition, a previously studied microbial Unlabeled subreads account Echinicola vietnamensis 1 100% Genus Strain Table 2 (Left): community from a lake known to predominantly contain poorly characterized phyla[1], and a for < 2.5% of the over 200,000 Escherichia coli 1 99.86% Family Species Analysis of the OTU consensus multiplexed. These results are compared to traditional 16S tag sequencing of the V4 hyper-variable high-quality subreads. The Fervidobacterium sequences generated by region with short-read sequencing technologies. We explore the benefits of using full-length dashed line represents the pennivorans 1 99.93% Genus Strain rDnaTools. Classification of the ribosomal DNA amplicons for estimating community structure and diversity, as well as for single-cell 500 subreads recommended Frateuria aurantia 1 99.93% Genus Strain Figure 4. Hirschia baltica 1 100% Genus Strain V4 region was carried out with isolate strain identification relative to alternate methods. We characterize the potential benefits of for phasing. A) Comparison of differences between two Salmonella spp. in the full-length 16S gene (2.6%) and Meiothermus silvanus 1 100% Genus Species the RDP Classifier[5] on V4 profiling metagenomic communities with full-length 16S rRNA genes from SMRT sequencing relative hyper-variable V4-region (0%) Olsenella uli 1 100% Genus Strain sequences extracted from the to standard methods. Pseudomonas stutzeri 1 100% Genus Strain Figure 7 (Left-Bottom): reference genomes. B) Example of the over-estimation of diversity that can result from classification based on small Salmonella bongori 2 99.93% Family Strain hyper-variability regions compared to the full-length sequence Histogram of consensus Salmonella enterica 1 99.24% Genus Serovar Classification of full-length 16S sequence counts by barcode. Segniliparus rotundus 1 99.93% Genus Strain was performed with RDP Multiple consensus sequences Spirochaeta smaragdinae 1 100% Genus Strain SeqMatch[5] on the OTU Streptococcus pyogenes 1 100% Genus Strain Reads-of-Insert Consensus enable better classification, consensus sequences Environmental Sample Terriglobus roseus 1 100% Genus Strain 0.1 generated by rDnaTools Thermobacillus composti 1 100% Genus Strain Community analysis of environmental Reads-of-Insert (RoI) Consensus Sequences 16S sequence data from Lake Sakinaw, B B B a B a B a c a c B c t a t e B c B t e a c e r ; t a B e r a c t i r a i 3 e c ; a t r i a c ; a ; e t r i F 9 ; C c t a e i I 9 e r ; C a F B t C i ; r h I F e r a C i G ; h I a a i ; C h l r a ; l o G h i a c C o ; a l ; G ; ; i h C o r ; t C l r a 9 e ; h o o l d i r o a C o h i i h r o r l f F o d o f l I i r l o i h e d l f l o a o i o r l e ; f c l o e x G ; o l r o r f o x ; 9 e c C o c l ; o f x i e c r i ; a o l x c F ; f 9 h o f e i D i c I x l ; D ; l i c e o D l f e x ; d F i e o o D i G I 9 l o ; e x c e x i l ; ; D e c r ; h o F o x i D e h i o G 9 a a I ; l ; h o e a c i ; D l ; f i D h a h a F ; e a c l h l d a G a i I 9 e D l o i a e ; e e h h l o o ; a o h d F x l c o a G h e h e c i I a o c i ; 9 l D i o e c o c a ; ; ; h a o o o l c d F i a D l G D o o c i c i c ; I a l ; D c 9 l c o x i o o ; a o o c c c d e o i c a ; c F l c i G e x i o c c c I c o c o h ; h c l x o 9 c o o f e d o c PacBio’s long read-length and circularized c o c o e o a i c i a c l e l i G F ; o i d c o f l o ; I o c d c c o i o l f D o c 9 British Columbia. Illumina V4-region r B d a d i i U l o U c a c c o ; i ; c U o d i a c B i i i G ; o o F ; a h a d i o r a ; d c o o o I o l c a ; c i ; r i n a n ; x l c d n d 9 c d a ; e v o i e o o i i h c a i d ; h l o a v o i r c c G d d c d e a o F t i c c ; v a e l l e l c o ; ; e i a ; D c i I t a e h d u v l d ; l f h i C e ; e a i l f i h ; c i t a 9 d o c v a a a i i r a a a d l ; i ; f o a d D i Conclusion G i f ; o e l r i i c v d C d c o ; o s F a a a i s x ; h ; i u ; s ; ; s ; C e r i a i d I d i n ; s d a a v a i i o c 2 F ; i n o c i i s e D ; ; d s s e e a s s f o l 9 d n r a i x ; i C a i B d l c ; U v s i e l h c o G 0 n d i i a i n B W f f C i s i i i F a i i a ; i f e n B f e r ; D d ; a f s e o o l f e c I − u h U d a h l x a A i i ; i n B i t a r i o l ; A i l i h n e s f h a e d i c 9 d B e l e s f ; c i ; c r l i 2 e ; A i o B U c e D a G e 2 t s l o n c n c C A s o l x e N B d d d 2 c t ; o o ; F o d a e d d i n ; o f c 3 A 2 s i r i l a a i I r l n 2 c l s l h c B 6 n e e n a a ; f c a r o ; n c c 2 6 D 2 a A ; e o l l s a i x a o i e r ; o Unclassified; c e o l B 6 U a a i h f ; 2 ; i B r G c A l l i o o f a s U a − c U p 6 ; f u 2 c a r e h l c d ; f l U n i t ; l 6 ; o l i l e s s A B c l C h o x D c 7 a l e o a 2 ; n e ss l r f ; e a c a U n 6 B s ; o i d x ; c i e u s i U t e o 1 x l s f 2 6 n C h o o l h l o e c ; a s a c d c i i U l ; l r f x D 1 n r ; U n − i i n c i ; c i i i s e 6 ; e a c n ; D l f U a r C e i l u D c a i a c l a h o o l o o S i t 4 i U i ; l h u U n f e d ; r f x D l o l e l s n c e r c d ; i B t C ; e c F l u e c a B e d ; a h o e i a ; e h s i ; o l c n n c e l r a c h U l s ; U t r f x h o a a a a i − d a C D l o i n a c f i ; h o ; s U a c e l o e i e a c C e l n l s i 6 ; t r r l d ; u ; B l o U a i e a C f x i c ; o c s f 5 B a c e i ; h D h lo s d c i d t r o o ; o a e a c n l s i e l e i e e e o U a f ; B a a C r l a c l e a r c o c s ; c e i ; h f x c i d t r o D h c a n e u t c n l s i e B a l o ; li t e f ; c a C e i e o e c l ; c c U a s i d e i r l r c B t ; h f c n o a u d i o o s i e a r o x i r a n l f ; c a l o D o l e c e i a s i d e i C r le ; l i e r ; id d U c e B t r ; h i n n C s i ; a f x d a li u i l f d c a o a l ;u lt ia a n a s i B e i C l o n o h e a t ; r e h a r e u ; ; c s i ; r h l A ; lo v f d c o f e C ; e a c d S l s i B e ia l ; r a a e ; a t C o s a e n sequence templates enable the generation of h U i r ; r D e o d s f d c h i; e n c u B f 7 i B e a o a l a ; data was analyzed with iTagger[2], while n s e ; a t i C l x A e le 6 in r ; a ; e lture a c i d c h e e a c x 5 B f B e a le n s n l ie ; a t i ; f i n li e cu te i; B a r C il li le c n D s A d B c ; d o o r U − e a r d o a r a ;u i e s 2 ; a t i ie o l r e e e a n A r f l e ; h if 6 B c i a n a in C a U c G i ; a e h a li n l l e t s ;C n o eae h l n a B c s C ; i ro A r l o s − d ; x A ; ac o c U c 1 ; a a d ; e s e r o l s l ia e e e a e a e o n a i 1 B c r i ; fl l n n f c s f 1 if a n a l c c ie n e d o A oli e U l s ; t s e r e ;A e ; r x o a i d U c s i o n n s i i n f if l li e li e e ; d c s ie ; a la a l D i U s B s h o o a na a la i d c s r e r B e ; n f n ;C ; e n e e a h N s ie ; la a li a n c a cl s U ia d li a d c r e n o n es;A te lo p a if n i r ro Ba ss i ; e if ; ;A e ;A al ri c o e U t s d i a e e e a o U l d c e x a ct ; c i− if ; a s i e n a n lin C c n ie a if ; l A e eria h o c 4 B l s d f i; n ;A ro l i U l B d c s e o x li e e ; o d a − ; n i r o 6 ;C r ia n s la if lo le r a a L o c s 6 U c s f e e n h fl ;S U l i 5 s h o a in A B lo e a fi ; n C r l S x h n s e U la ; o n o e; rofle i 7 c s d c a l A r ;M ;D 6 l i ; ri h i; e ea a 5 a fi n C x a ii e s e U te ; ; n lin r xi h B s d c a le o ; ;De a − i ; i d f ;A 6 high-quality consensus sequences from l U A f a r e o i cte L o ie B e fi r x aer a B PacBio full-length 16S data was c n G d t i lo e b hal c s l S B o c − ; a s h f a c la 1 o so ;M c oc c s B la ;C r u a t o s 1 c a lo exi;An F ii e o id i 1 n ri h l r ri c i fi ; ia; te a c a e U te ;C rof r c ;C oid ;G d c a e a h ; a ri lo b lo ia;G IF B e act o r Unclass 3 t b s o c a;Ch o u fle ; a i ;F x IF3 B er us a i U t ;F ri ;D n ; c e e c ifi a ia ; ct h la ed; B ter a al U s c ified b o n s a s o • The PacBio RSII enables high-throughput sequencing of full- c c i s o l fie B u c a d F c U s ; clas ; oi n si n ia ; d cl fi U r d ia a ed te ie ;v s ; c if a si a s ; d fie B s d in d la ie U B ; nc if n A U ss ; 1; cl 2 a d 1 a 6; cl ie P U ss n if O n i U s multiple passes over the same molecule.
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