次世代シーケンサーを用いた解析のご紹介<16S …

1 DNA 16S rRNA PCR PCR 1-5 g GS FLX 1 16S rRNA RDP Classifier(1) <16S rRNA > 1 2 3 16S rRNA 5 Domain Genus NoTaxo nCo u n t1 Nocardiopsaceae56692 Bacillaceae13243 Actinomycetales8924 Glycomyces5275 Flavobacteriaceae4756 Yaniella4537 Brachybacterium3368 Bacilli3199 Micrococcineae27810 Aequorivita19811 Bacillales15312 Gammaproteobacteria14913 Planifilum14814 Streptosporangineae12415 Brevibacterium11416 Unclassified_Bacteria9217 Pseudonocardiaceae9118 Arthrobacter8019 Corynebacterium7020 Joostella61

2 Domain Genus Rae d_ n ameDomainB ootstrapvalu ePh ylu mB ootstrapvalu eClassB ootstrapvalu eSu bc lassB ootstrapvalu eOrde rB ootstrapvalu eSu borde rB ootstrapvalu eFamilyB ootstrapvalu eGe n u sB ootstrapvalu Burkholderiales1 Chart of abundance of the taxa found by RDP classifier NocardiopsaceaeBacillaceaeActinomycetale sGlycomycesFlavobacteriaceaeYaniellaBrac hybacteriumBacilliMicrococcineaeAequoriv itaBacillalesGammaproteobacteriaPlanifil umStreptosporangineaeBrevibacteriumUncla ssified_BacteriaPseudonocardiaceaeArthro bacterCorynebacteriumJoostella Qu e ryQUERYSIZEQUERYHITC omme n tORGC omme n t2Le n gthHitPosNoSc oreExpe c tIde n titie s_ 1 Ide n titie s_ 2 GapsStran dQUERYSTARTQUERYENDSUB JECTSTARTSUB JECTENDQUERYSTRINGSUB JECTSTRINGFG7DF1X10F6 QXY length=120 xy=2420_0340 region=10run=R_2008_09_17_16_58_39_1201E F010836_1|Virgibacillus sp.

3 (h)-06|16S ribosomal RNAV irgibacillus sp. (h) length=120 xy=2420_0340 region=10run=R_2008_09_17_16_58_39_1202E U037266_1|Bacillus sp. G1DM-84|16S ribosomal RNAB acillus sp. length=120 xy=2420_0340 region=10run=R_2008_09_17_16_58_39_1203E F623842_1|Bacillus cereus|16S ribosomal RNAB acillus length=120 xy=2420_0340 region=10run=R_2008_09_17_16_58_39_1204E F113684_1|Bacillus thuringiensis|16S ribosomal RNAB acillus length=120 xy=2420_0340 region=10run=R_2008_09_17_16_58_39_1205E F101166_1|Virgibacillus sp. 30-7|16S ribosomal RNAV irgibacillus sp. length=121 xy=2439_0368 region=10run=R_2008_09_17_16_58_39_1211E U274333_1|Micromonospora sp.

4 QAII13|16S ribosomal RNAM icromonospora sp. length=121 xy=2439_0368 region=10run=R_2008_09_17_16_58_39_1212E F216369_1|Knoellia sp. TFS 104|16S ribosomal RNAK noellia sp. TFS length=121 xy=2439_0368 region=10run=R_2008_09_17_16_58_39_1213A F060684_1|Kineococcus-like bacterium AS3088|16S ribosomal RNAK ineococcus-like bacterium length=121 xy=2439_0368 region=10run=R_2008_09_17_16_58_39_1214F J482120_1|bacterium SRU_17|16S ribosomal RNAbacterium length=121 xy=2439_0368 region=10run=R_2008_09_17_16_58_39_1215A F060675_1|Kineococcus-like bacterium AS2965|16S ribosomal RNAK ineococcus-like bacterium * 1 Wang Q et al., (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.

5 Appl. Environ. Microbiol. 73(16):5261-7 FLX adaptor A FLX adaptor B tag 1 2 3 4 1 DNA PCR primer F primer R 2 PCR 3 Domain Genus Escherichia Enterobacteriaceae Species Strain <16S rRNA UniFrac(1) > Unifrac distance = ( [ + ]) / ( [ + + ]) 0 0 0 PCoA (Principal Coordinates Analysis ) Cluster Samples Weighted Unifrac : community structure ( ) Unweighted Unifrac : community member ( ) Unifrac Distance Matrix * 1 Hamady M et al.

6 , (2010) Fast UniFrac: facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and PhyloChip data. ISME Journal 4(1): 17 27 2 Turque AS et al., (2010) Environmental shaping of sponge associated archaeal communities. PLoS One. 30;5(12):e15774. Turque (2) 2 Accession No: GU058339-GU058920 1 2 3 Input data1 Environment file* Input data 2 4 Input data 1 2 Unifrac distance matrix Input data 1 Sample1 Sample2 Sample3 Read1 Sample1 100 Read2 Sample1 50 Read3 Sample2 200 Read4 Sample3 40 Read5 Sample3 30 Input data 2 Environment file vs vs vs Unifrac Distance Weighted Unweighted * Sample1 Sample2

7 Sample1 Sample2 Sample1 Sample2 Unifrac Distance Cluster Samples PCoA 1 2 3 UPGMA Unweigted Pair Group Method with Arithmetic mean: Unifrac distance = PCoA Scatter Plot Cluster Samples HhCA Cagarras * Hymeniacidon heliophila HhP92 Guanabara * Hymeniacidon heliophila PcCA Cagarras Petromica citrina PmCA Cagarras Paraleucilla magna PmP92 Guanabara Paraleucilla magna SwCA Cagarras free-living SwP92 Guanabara free-living * 1, Cagarras * 2.

8 Guanabara Hh CAHh P9 2Pc CAPmCAPmP9 2 SwCASwP9 2Hh P9 Unifrac distance matrix Unweighted Cagarras Guanabara HhCA PmCA PcCA HhP92 PmP92 free-living Turque -Percent variation explained P1 -Percent variation explained PCoA -P1 vs P2 HhCAHhP92 PcCAPmCAPmP92 SwCASwP92 Cagarras Guanabara