greengenes.lbl.govgreengenes.lbl.gov 16S rRNA gene database 16S rRNA gene database

and workbench compatible and workbench compatible with ARB with ARB

Todd DeSantis, Phil Hugenholtz, Niels Larson, Igor Dubosarskiy, Jordan Moberg, Yvette Piceno, Ingrid

Zubieta, Eoin Brodie, Gary Andersen

LBL - JGI

Andersen Group Program Aims

• Creating a microarray for the simultaneous differentiation and quantification of closely related prokaryotes in complex samples.

The Biomarker

16S rDNA

rRNA (functional molecule)

LSU

SSU

16S rDNA - identify and classify organisms by gene sequence variations.

The Challenges• 16S sequence deposit rate is increasing.• Many are mis-annotated and/or chimeric.• Sequence Taxonomy updates lags years

behind sequence availability (“Bacteria, Unclassified”).

• Difficult to create and manage MSAs of all 16S seq data (or even thousands) using Clustal/BioEdit/Arb.

• Probe quality is reliant on excellent MSAs and taxonomy.

• “Signatures” can erode as more sequences are discovered.

greengenes.lbl.gov

greengenes.lbl.gov

Stay current

020,00040,00060,00080,000

100,000120,000140,000160,000180,000200,000

Cum

ulativ

e N

CBI

16S

record

s

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

Year

Source: http://www.ncbi.nlm.nih.gov/

‘16S NOT 1.16S NOTmitochondr* NOT 18S’

Fate of NCBI Records:

short FASTA file (9%)

short BLAST match length (8%)

BLAST match to 18S/Mito SSU (1%)

odd nt insertions (1%)

passed (81%)

greengenes.lbl.gov

Verify ‘16S-ness’

NAST alignstep 1: find template

• Hand curated MSA provided by Phil.

• Alignment "template" is top BLAST HSP– q= -1, Favors long

match• Candidate trimmed

of extra-16S seq data– tRNA, intergenic

spacer regions, and 23S rDNA

– based on HSP boundries

• If HSP paired opposite strands, candidate is reverse complemented.

NAST alignstep 1: find template

• Hand curated MSA provided by Phil.

• Alignment "template" is top BLAST HSP– q= -1, Favors long

match• Candidate trimmed

of extra-16S seq data– tRNA, intergenic

spacer regions, and 23S rDNA

– based on HSP boundries

• If HSP paired opposite strands, candidate is reverse complemented.

NAST alignstep 1: find template

• Hand curated MSA provided by Phil.

• Alignment "template" is top BLAST HSP– q= -1, Favors long

match• Candidate trimmed

of extra-16S seq data– tRNA, intergenic

spacer regions, and 23S rDNA

– based on HSP boundries

• If HSP paired opposite strands, candidate is reverse complemented.

NAST alignstep 1: find template

• Hand curated MSA provided by Phil.

• Alignment "template" is top BLAST HSP– q= -1, Favors long

match• Candidate trimmed

of extra-16S seq data– tRNA, intergenic

spacer regions, and 23S rDNA

– based on HSP boundries

• If HSP paired opposite strands, candidate is reverse complemented.

NAST alignstep 2: gap removal

Preserves global MSA positions(columns) by allowing local misalignments.

DEFINESt = post-Align0 template sequence.Sc = post-Align0 candidate sequence.Ht = alignment space (hyphen) inserted into St by

Align0.Hc = alignment space (hyphen) inserted into Sc by

Align0. WHILE (St contains one or more Ht) DO

LHt = character index of distal 5' Ht within St

L5' = character index of Hc within Sc which is 5' proximal to Ht

L3' = character index of Hc within Sc which is 3' proximal to Ht

IF ((LHt – L5') > (L3' – LHt)) Delete Hc found at L3'

ELSE Delete Hc found at L5'Delete template gap character.

END WHILE

Result: Largest MSA of full-length (>1250 nt) 16S rDNA genes.

Isolate tag present?

“Genus species” style name in DEFINITION or

source>organism?

Is sequence from whole genome

record?

Genbank record

yes

Record is from an isolateyesno

Glob text from “DEFINITION”,

“source”, and “TITLE”

no

Text glob

“Gs yes”

Text glob contains “clone” OR “uncultur”?

Record is from a clone

Text glob contains “symbiont”?

Record is from a symbiont

yes no

noyes

Does a source>isolate

field exist?

yesno

Record is from undecided

“Gs no”

yesno

“Isolate tag no”

“Isolate tag yes”

Gs result? if Gs

yes

no

Strain tag is present

Record is from a isolate_str

greengenes.lbl.gov

Name generator• NCBI annotations are non-standardized

– Determine if sequence is from an isolate, environmental amplicon/metagenome

– Concatenate useful terms

• Effort to guide future GenBank submitters in clear record descriptions – http://www.jgi.doe.gov/16s/

greengenes.lbl.gov

Chimera tracking• Amplicons from complex gDNA can

contain partial sequence from more than one genome.

• Up to 4% of sequences are deemed chimeric by Bellerophon2– Flags are set to avoid using these

questionable sequences in phylogeny assessments

greengenes.lbl.gov

Maintain TaxonomyJGI taxonomy organized in ARB using maximum parsimony tree insertions.Example: http://greengenes.lbl.gov/cgi

-bin/User/show_one_record_v2.pl?prokMSA_id=82172

prokMSA_id: 82172 prokMSAname: termite gut clone Rs-050

GenBank ACCESSION: AB100461.1, GenBank GI: 28971862, RDP_id: S000122947, NCBI_tax_id: 203524, Study_id: 21358

G2_chip_tax_string=Bacteria; Firmicutes; Clostridia; Clostridiales; Peptostreptococcaceae; sf_5; otu_2988

JGI_tax_string=Bacteria; Firmicutes (incl. basal lineag; Firmicutes; Peptostreptococcaceae; Mogibacterium

JGI_tax_string_format_2=Bacteria; Firmicutes (incl. basal lineag; Firmicutes; Peptostreptococcaceae; Mogibacterium; otu_415

Pace_tax_string=Bacteria; Firmicutes; Clostridium et al.; Peptostreptococcaceae; Clostridium acidiurici et al.; Clostridium difficile et al.; Clostridium aminobutyricum et

RDP_tax_string= Bacteria; Firmicutes; Clostridia; Clostridiales; unclassified_Clostridiales.

ncbi_tax_string=Bacteria; Firmicutes; Clostridia; Clostridiales; Eubacteriaceae; environmental samples

greengenes.lbl.gov

Maintain Taxonomy

greengenes.lbl.gov Tools

• BLAST• SimRank• Probe matcher• Text search• PCR primer design• Private NAST aligner

greengenes.lbl.gov Compatible with ARB

• Entire data base download-able in ARB format.

• Can import new records into personal ARB data base.

How we use greengenes data to get our work

done…..

16S Sequence clustering

• Each sequence reduced to an array (list) of “probe-friendly” 25-mers which:– Have high complexity– Can be synthesized with 75 or fewer masks– Adequate H-bond potential

• G+C content over 48%• Or empirical bond stability found in test arrays

• Transitive clustering by fraction of 25mers in common – Cluster considered an Operational Taxonomic

Unit (OTU)

Extended Bergey’s TaxonomyBergey’s v0.9 with added nomenclature from

Hugenholtz tree of environmental DNA• Each OTU assigned to one of 455 families• Families split into subfamilies where >15%

sequence variation existed. • Results: (considering both domains)

• 63 phyla• 136 classes• 262 orders• 455 families• 842 subfamilies (~94% identity)• 8,989 OTUs (~99% identity)• 30,627 sequences (each belong to only one OTU)

Bacteria; Proteobacteria; Deltaproteobacteria; Desulfovibrionales; Desulfovibrionaceae; sf_1; otu_10051

Desulfovibrio sp. str. DMB.Desulfovibrio sp. 'Bendigo A'Desulfovibrio vulgaris DSM 644

Regions unique to OTU

Regions not unique to OTU Sequence discrepancies

Example of the Location of Probes Used for the Desulfovibrio vulgaris Probe Set

Probe Design

Locus Specific Prevalence ScoringExample: proteobacteria

OTU composed of 26 sequences

22/22 25/25 20/25

Probe selection objectives for each OTU

• Find 11 or more 25mers (targets) – >90% prevalent in an OTU’s sequences– dissimilar from sequences outside the OTU– >48% G+C or empirically responsive– >1 loci within 16S rDNA gene

• Presumed cross-hybridizing probes were those 25-mers that contained a central 17-mer matching sequences in more than one OTU (Urakawa, Stahl et al. 2002)– avoiding probes that were unique solely due to a mismatch in

one of the outer four bases. • As each PM probe (Perfect Match to target) was chosen, it was

paired with a control 25-mer (mismatching probe, MM), identical in all positions except the thirteenth base.

• The MM probe did not contain an internal 17-mer complimentary to sequences in any OTU.

18 µ

18 µ

AC

GG

TC

GA

AC

GG

TC

GA

AC

GG

TC

GA

AC

GG

TC

GA

AC

GG

TC

GA

Hybridize

PCR Amplify DNA

Fractionate DNA

End-label with biotin

Extract Genomic DNA

Overview of Sample Preparation

Image Capture and

Data Reduction

•Over 500,000 data points

SUBG

ROUP

desc

riptio

n

81_L

os_A

lamos

.CEL

81_U

ltra_S

oil.CE

L

84_L

os_A

lamos

.CEL

84_M

_Mille

r.CEL

90-Lo

s_Ala

mos.C

EL

90-P

ower_

Soil.C

EL

Airpo

rt_1.C

EL

Airpo

rt_2.C

EL

Airpo

rt_6.C

EL

Airpo

rt_7.C

EL

Airpo

rt_A_

1.CEL

Airpo

rt_A_

2.CEL

Airpo

rt_B_

1.CEL

Airpo

rt_B_

2.CEL

COUN

TIF pf

==1

'022102 CHLOROPLASTS_AND_CYANELLES 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14'02280110 SPHINGOMONAS_GROUP 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14'0228030406MCH.PURPURATUM_SUBGROUP 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14'021506 CY.AURANTIACA_GROUP 1 1 1 1 1 1 1 1 1 0.94 1 1 1 1 13'0228010616MSO.LOTI_SUBGROUP 1 1 1 1 1 1 1 1 1 0.94 1 1 1 1 13'02280313 PSEUDOMONAS_AND_RELATIVES 0.96 1 1 1 1 1 1 1 1 1 1 1 1 1 13'0230011302CORYNEBACTERIUM_GROUP 1 1 0.94 1 1 1 1 1 1 1 1 1 1 1 13'021304 ENVIRONMENTAL_CLONE_OPB5_GROUP1 1 0.91 1 1 1 1 1 1 1 1 1 1 1 13'02150402 FLX.SANCTI_SUBGROUP 1 1 1 1 1 1 1 1 1 0.94 1 1 1 1 13'0228010608BL.VIRIDIS_ASSEMBLAGE 1 1 1 1 1 1 1 1 1 0.91 1 1 1 1 13'02280211 OXALOBACTER_GROUP 1 1 0.89 1 1 1 1 1 1 1 1 1 1 1 13'02280308 XANTHOMONAS_GROUP 1 1 1 1 1 1 1 1 1 0.92 1 1 1 1 13'0230010901ARTHROBACTER_AND_RELATIVES 1 1 0.92 1 1 1 1 1 1 1 1 1 1 1 13'02300110 PROPIONIBACTERIUM_GROUP 0.9 1 1 1 1 1 1 1 1 1 1 1 1 1 13'0218 ENVIRONMENTAL_CLONE_WCHB1-31_GROUP0.94 1 0.89 1 1 1 1 1 1 1 1 1 1 1 12'02250306 ACBT.CAPSULATUM_GROUP 1 1 1 1 1 1 1 1 0.96 0.92 1 1 1 1 12'0228010611METHYLOBACTERIA_SUBGROUP 0.95 1 1 1 1 1 1 1 1 0.91 1 1 1 1 12'022801061210BDR.ELKANII_SUBGROUP 1 1 1 1 1 1 1 1 0.98 0.67 1 1 1 1 12'022801061214BLB.DENITRIFICANS_SUBGROUP 1 1 1 1 1 1 1 1 1 0.79 1 0.95 1 1 12'022802090401COM.TERRIGENA_SUBGROUP 0.95 1 0.7 1 1 1 1 1 1 1 1 1 1 1 12'02300710 B.MEGATERIUM_GROUP 0.94 1 0.93 1 1 1 1 1 1 1 1 1 1 1 12'02300901 C.LEPTUM_GROUP 0.89 1 0.94 1 1 1 1 1 1 1 1 1 1 1 12'022801080102PARACOCCUS_SUBGROUP 0.91 1 1 1 1 0.91 1 1 1 1 1 1 1 1 12'0228040603POL.CELLULOSUM_SUBGROUP 0.82 1 1 1 1 1 1 1 1 0.82 1 1 1 1 12'02280108010101ROS.DENITRIFICANS_SUBGROUP 0.95 1 1 1 1 0.95 1 1 1 0.95 1 1 1 1 11'0228050301AOB.CRYAEROPHILUS_SUBGROUP0.95 1 0.95 1 1 1 1 1 1 1 1 0.95 1 1 11'023001130101MYB.TUBERCULOSIS_SUBGROUP 0.94 1 1 1 1 0.94 1 1 1 0.95 1 1 1 1 11'0228010404AZS.LIPOFERUM_SUBGROUP 0.96 1 0.96 1 1 1 1 1 1 0.93 1 1 1 1 11'022801061201AFIPIA.FELIS_SUBGROUP 1 1 1 1 1 1 1 1 0.98 0.89 1 0.95 1 1 11'022801061204NTB.WINOGRADSKYI_SUBGROUP 1 1 1 1 1 1 1 1 0.94 0.78 1 0.94 1 1 11'022801061205RPS.PALUSTRIS_SUBGROUP 1 1 1 1 1 1 1 1 0.97 0.87 1 0.96 1 1 11'022801061208BDR.LUPINI_SUBGROUP 1 1 1 1 1 1 1 1 0.96 0.71 1 0.96 1 1 11'022801061212BDR.LIAONINGENSIS_SUBGROUP 1 1 1 1 1 1 1 1 0.95 0.79 1 0.95 1 1 11'0228020403NSS.MULTIFORMIS_SUBGROUP 0.96 1 0.96 1 1 1 1 1 1 0.87 1 1 1 1 11'021306 ENVIRONMENTAL_CLONE_III1-8_GROUP0.93 1 1 1 1 1 1 1 0.97 0.85 1 1 1 1 11'02200101 PIRELLULA_SCHLESNER_ISOLATES0.92 1 0.92 1 1 1 1 1 1 0.94 1 1 1 1 11'02280410 DESULFOBULBUS_ASSEMBLAGE 0.92 1 1 1 1 1 1 1 1 0.89 1 0.95 1 1 11'02300711 B.SUBTILIS_GROUP 0.85 1 0.67 1 1 1 1 1 0.97 1 1 1 1 1 11'021505 PERSICOBACTER_GROUP 1 1 1 1 1 1 1 1 0.86 0.82 1 1 1 0.92 11'022804010401DSV.HALOPHILUS_SUBGROUP 0.5 1 0.42 1 1 0.92 1 1 1 1 1 1 1 1 11'0230011201PSC.HALOPHOBICA_SUBGROUP 0.92 1 1 1 1 0.92 1 1 1 0.93 1 1 1 1 11'0230040105BTV.FIBRISOLVENS_SUBGROUP 0.68 1 0.78 1 1 0.84 1 1 1 1 1 1 1 1 11'02280327 ENTERICS_AND_RELATIVES 0.91 1 0.86 1 1 1 1 1 0.99 0.98 1 1 1 1 10'0230010602A.FERROOXIDANS_SUBGROUP 0.96 1 0.96 1 1 0.94 1 1 1 0.91 1 1 1 1 10'02250301 MOUNT_COOT-THA_ENVIRONMENTAL_CLONES_III0.89 1 0.89 1 1 0.9 1 1 1 1 1 0.96 1 1 10'0228010609MSI.TRICHOSPORIUM_SUBGROUP 0.77 1 0.87 1 1 0.96 1 1 1 0.78 1 1 1 1 10'0228020804BRD.BRONCHISEPTICA_SUBGROUP0.82 1 0.91 1 1 1 1 1 0.97 0.77 1 1 1 1 10'02300111 MICROMONOSPORA_GROUP 0.83 1 0.83 1 1 0.83 1 1 1 0.94 1 1 1 1 10'0230070903B.ALCALOPHILUS_SUBGROUP 0.81 1 0.46 1 1 0.88 1 1 1 0.98 1 1 1 1 10'0205 ENVIRONMENTAL_CLONE_OPB45_GROUP0.73 1 0.91 1 1 1 1 1 0.91 0.6 1 1 1 1 10'021312 ENVIRONMENTAL_CLONE_RB40_GROUP0.82 1 0.81 1 1 0.8 1 1 1 0.74 1 1 1 1 10'0215010204CY.FERMENTANS_SUBGROUP 0.94 1 1 1 1 0.94 1 1 0.92 0.91 1 1 1 1 10'02280108010105OCT.ANTARCTICUS_SUBGROUP 0.71 1 0.75 1 1 0.73 1 1 1 0.64 1 1 1 1 10'023001080110THERMOPHILIC_STREPTOMYCES 0.75 1 0.8 1 1 0.88 1 1 1 0.88 1 1 1 1 10'0230040103EUB.SABURREUM_SUBGROUP 0.4 1 0.53 1 1 0.75 1 1 1 0.56 1 1 1 1 10'02300713 B.SPHAERICUS_GROUP 0.86 1 0.71 1 1 0.92 1 1 1 0.93 1 1 1 1 10'0230072109STC.PNEUMONIAE_SUBGROUP 0.92 1 0.92 1 1 1 1 1 1 0.85 1 1 1 0.93 10'02250102 ENVIRONMENTAL_CLONE_OCS307_GROUP0.89 1 1 1 1 0.94 1 1 1 1 0.94 0.94 1 0.94 9'0230040104RUC.GNAVUS_SUBGROUP 0.9 1 0.95 1 1 0.96 1 1 0.96 0.95 1 1 1 1 9'021305 ENVIRONMENTAL_CLONE_RB25_GROUP0.93 1 0.95 1 1 1 0.95 1 0.96 0.83 1 1 1 1 9•Scores for each of 9000 OTUS

Distribution of 16S rDNA Sequences detected Distribution of 16S rDNA Sequences detected via Cloning or Microarray Analysisvia Cloning or Microarray Analysis

Clone Hits Only (8)

Clone and ArrayHits (73) Array Hits

Only (97)

Confirmed by specific PCR and sequencing:Actinobacteria; Actinosynnemataceae; sf_1Nitrospira; Nitrospiraceae; sf_1Clostridia; Syntrophomonadaceae; sf_5Planctomycetes; Plantomycetaceae; sf_3Gammaproteobacteria; Pseudoaltermonadaceae; sf_1Acidobacteria; Ellin6075/11-25; sf_1Spirochaetes; Spirochaetaceae; sf_1Spirochaetes; Spirochaetaceae; sf_3Spirochaetes; Leptospiracea; sf_3

6

7

8

9

10

11

12

13

14

1 2 3 4 5 6 7 8

log2 Concentration (pM)

log 2

Hyb

Sco

re (

a.u.

) r = 0.917

Spike–in% G+C

sequence % G+C probes

Mycoplasma neurolyticum

50.0 45.4

Oenococcus oeni 50.9 50.8

Saprospira grandis 51.8 50.9

Fervidobacterium nodosum

58.2 53.8

Caulobacter vibrioides

56.4 58.5

Array is quantitative

Array is quantitative

~107 16S gene copies

~1011 16S gene copies

Example query against meteorological data:

Does detection of Actinobacterium PENDANT-38 correlate with temperature?

r = 0.64, p=0.026527(adjusted for multiple testing)

4

4.5

5

5.5

6

6.5

75 80 85 90

Temp. degC

log(H

ybSc

ore

)

Real-time quantitative PCR confirmation of array monitoring. Real-time quantitative PCR confirmation of array monitoring.

Representative organism Phylocode GroupCorrected Array Intensity

Area 2 Reduction Oxidation

Geothrix fermentans 2.13.8.386 Acidobacteriaceae 45 2344 2290

Geobacter metallireducens 2.28.4.7.4.10207 Geobacteraceae 251 2238 2188

Geobacter arculus 2.28.4.7.4.10209 Geobacteraceae 38 1412 1698

Species specific - Geothrix fermentans Group specific - Geobacteraceae

(a) Array quantitation

(b) qPCR quantitation

Uranium BioremediationUranium Bioremediation – is uranium re-oxidation under reducing conditions due to loss of metal reducers? – is uranium re-oxidation under reducing conditions due to loss of metal reducers?

Real-time quantitative PCR confirmation – Urban AerosolReal-time quantitative PCR confirmation – Urban Aerosol

Array hybridization signal correlates significantlyArray hybridization signal correlates significantlywith 16S copies in environmental aerosol DNA extractwith 16S copies in environmental aerosol DNA extract

Pseudomonas oleovoransPseudomonas oleovorans example example

Order Class Peak Duration (sec)

Phaeophyceae (phylum) Stramenopiles (no rank) 5

Basidiomycota (phylum) Fungi (kingdom) 45

Deferribacterales Cyanobacteria 450

Ascomycota (phylum) Fungi (kingdom) 450

Vibrionales Gammaproteobacteria 450

Flavobacteriales Flavobacteria 450

Clostridiales Clostridia 45

Rhizobiales Alphaproteobacteria 45

Rhodospirillales Alphaproteobacteria 45 n.s.

Lactobacillales Bacilli 45

Bacillales Bacilli 450

Mycoplasmatales Mollicutes 5

Xanthomonadales Gammaproteobacteria 5 n.s.

Burkholderiales Betaproteobacteria 0

Sphingomonadales Alphaproteobacteria 0

Sphingobacteriales Sphingobacteria 0

Acholeplasmatales Mollicutes 45

FEMS Letters - pseudoshift

Acknowledgements

• Phil Hugenholtz – Taxonomy, Arb Interface, Chimera

• Niels Larson – SimRank• Igor Dubosarskiy – JSP• Jordan Moberg – Microarrays, Cloning • Yvette Piceno – Microarrays, Primer Design• Ingrid Zubieta – PCR, Cloning• Eoin Brodie – Microarrays, QPCR• Gary Andersen – 16S Microarray Group Leader

...CGTAAAGCTCTGTCTTTGGGGAAGATAATGACGGTACCCAAGGAGGAAGCCACGGCTAACT... C. perf. str.CPN50

................................................................... C. perf. resistant

................................................................... Clostridium sp. AB&J

................................................................... clone p-4636-2Wa2

................................................................... C. perf. A

................................................................... C. perf rrnA

................................................................... C. perf rrnE

.................................T................................. C. perf rrnD

................................................................... C. perf rrnC

................................................................... C. perf rrnB

................................................................... C. perf rrnF

................................................................... C. perf rrnG

................................................................... C. perf str.13a

................................................................... C. perf str.13b

................................................................... C. perf rrnH

................................................................... C. perf rrnI

................................................................... C. perf rrnJ

................................................................... clone OI1612

................................................................... C. perf. B

................................................................... Swine manure 37-3

................................................................... Swine manure 37-4

TAAAGCTCTGTCTTTGGGGAAGATA tacccaaggaggaagccacggctaa AAAGCTCTGTCTTTGGGGAAGATAA AAGCTCTGTCTTTGGGGAAGATAAT AGCTCTGTCTTTGGGGAAGATAATG

Bacteria

CFB

Cyan

Proteo

Gram +

High G+C

Bacil-Strep

Clostridium

C.BOTULINUM_SUBGROUP

C.THERMOBUTYRICUM_SUBGROUP

C.BARATI_SUBGROUP

C.CADAVERIS

C.ALGIDICARNIS

C.PERFRINGENS

C.AURANTIBUTYRICUM

C. BUTYRICUM

16S rDNA

Probe Properties:25mer exits in 90% of the taxon’s seqsInternal 21mer exists only in one taxon.Probes 5 - 8

5 6 7 8

27 1492

420 469

Ave Diff =1891

C. perfringens probe set identified in EPA sample 22 (N.Y. Spring)