1. greengenes.lbl.gov 16S rRNA gene database and workbench compatible with ARB
Todd DeSantis, Phil Hugenholtz, Niels Larson, Igor Dubosarskiy, Jordan Moberg, Yvette Piceno, Ingrid Zubieta, Eoin Brodie, Gary Andersen
LBL - JGI

2. Andersen Group Program Aims
Creating a microarray for the simultaneous differentiation and quantification of closely related prokaryotes in complex samples.

3. The Biomarker
16S rDNA - identify and classify organisms by gene sequence variations.
16S rDNA
rRNA (functional molecule)
LSU
SSU

4. 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.

5. greengenes.lbl.gov

6. greengenes.lbl.gov Stay current
Source:
‘16S NOT 1.16S NOTmitochondr* NOT 18S’

7. greengenes.lbl.gov Verify ‘16S-ness’
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%)

8. NAST align step 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 align step 1: find template

9. NAST align step 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 align step 1: find template

10. NAST align step 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 align step 1: find template

11. NAST align step 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 align step 1: find template

12. NAST align step 2: gap removal
Preserves global MSA positions(columns) by allowing local misalignments.
DEFINE
St = 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.

13. greengenes.lbl.gov Name generator
Genbank record
Is sequence from whole genome record?
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 no
Glob text from “DEFINITION”, “source”, and “TITLE”
“Genus species” style name in DEFINITION or source>organism?
Does a source>isolate field exist?
Text glob contains “clone” OR “uncultur”?
yes
yes no
yes no
yes
Record is from an isolate no
if Gs
Gs result?
“Gs yes”
Text glob
“Isolate tag no”
“Isolate tag yes”
“Gs no”
yes no
yes no
Text glob contains “symbiont”?
Strain tag is present
Record is from a clone
Isolate tag present?
Record is from a symbiont
Record is from undecided
yes no
Record is from a isolate_str

14. 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

15. greengenes.lbl.gov Maintain Taxonomy
JGI taxonomy organized in ARB using maximum parsimony tree insertions.
Example:
prokMSA_id: prokMSAname: termite gut clone Rs-050
GenBank ACCESSION: AB , GenBank GI: , RDP_id: S , NCBI_tax_id: , 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

16. greengenes.lbl.gov Maintain Taxonomy

17. greengenes.lbl.gov Tools
BLAST
SimRank
Probe matcher
Text search
PCR primer design
Private NAST aligner

18. greengenes.lbl.gov Compatible with ARB
Entire data base download-able in ARB format.
Can import new records into personal ARB data base.

19. How we use greengenes data to get our work done…..

20. 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)

21. Extended Bergey’s Taxonomy
Bergey’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)

22. Probe Design Example of the Location of Probes Used for
Desulfovibrio sp. str. DMB.
Desulfovibrio sp. 'Bendigo A'
Desulfovibrio vulgaris DSM 644
Example of the Location of Probes Used for
the Desulfovibrio vulgaris Probe Set
Sequence discrepancies
Regions not unique to OTU
Bacteria; Proteobacteria; Deltaproteobacteria; Desulfovibrionales; Desulfovibrionaceae; sf_1; otu_10051
Regions unique to OTU

23. Locus Specific Prevalence Scoring
22/22
25/25
20/25
Example: proteobacteria OTU composed of 26 sequences
Locus Specific Prevalence Scoring

24. 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.

25. Overview of Sample PreparationC G T A C G T A C G T A C G T A C G T
Extract Genomic DNA
PCR Amplify DNA
18 µ
Fractionate DNA
18 µ
End-label with biotin
Hybridize

26. Image Capture and Data Reduction
Over 500,000 data points
Image Capture and Data Reduction
Scores for each of 9000 OTUS

27. Distribution of 16S rDNA Sequences detected via Cloning or Microarray Analysis
Clone Hits
Only (8)
Clone and Array
Hits (73)
Array Hits
Only (97)
Confirmed by specific PCR and sequencing:
Actinobacteria; Actinosynnemataceae; sf_1
Nitrospira; Nitrospiraceae; sf_1
Clostridia; Syntrophomonadaceae; sf_5
Planctomycetes; Plantomycetaceae; sf_3
Gammaproteobacteria; Pseudoaltermonadaceae; sf_1
Acidobacteria; Ellin6075/11-25; sf_1
Spirochaetes; Spirochaetaceae; sf_1
Spirochaetes; Spirochaetaceae; sf_3
Spirochaetes; Leptospiracea; sf_3

28. Array is quantitative 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
Fervidobacterium nodosum
58.2
53.8
Caulobacter vibrioides
56.4
58.5

29. Array is quantitative
~ S gene copies
~107 16S gene copies

30. Example query against meteorological data: Does detection of Actinobacterium PENDANT-38 correlate with temperature?

31. Real-time quantitative PCR confirmation of array monitoring.
Uranium Bioremediation – is uranium re-oxidation under reducing conditions due to loss of metal reducers?
(a) Array quantitation
Representative organism
Phylocode
Group
Corrected Array Intensity
Area Reduction Oxidation
Geothrix fermentans
Acidobacteriaceae 45
2344
2290
Geobacter metallireducens
Geobacteraceae
251
2238
2188
Geobacter arculus 38
1412
1698
(b) qPCR quantitation
Species specific - Geothrix fermentans
Group specific - Geobacteraceae

32. Real-time quantitative PCR confirmation – Urban Aerosol
Array hybridization signal correlates significantly
with 16S copies in environmental aerosol DNA extract
Pseudomonas oleovorans example

33. FEMS Letters - pseudoshift
Order
Class
Peak Duration (sec)
Phaeophyceae (phylum)
Stramenopiles (no rank) 5
Basidiomycota (phylum)
Fungi (kingdom) 45
Deferribacterales
Cyanobacteria
450
Ascomycota (phylum)
Vibrionales
Gammaproteobacteria
Flavobacteriales
Flavobacteria
Clostridiales
Clostridia
Rhizobiales
Alphaproteobacteria
Rhodospirillales
n.s.
Lactobacillales
Bacilli
Bacillales
Mycoplasmatales
Mollicutes
Xanthomonadales
Burkholderiales
Betaproteobacteria
Sphingomonadales
Sphingobacteriales
Sphingobacteria
Acholeplasmatales

34. 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

35. C. perfringens probe set identified in EPA sample 22 (N.Y. Spring)
CFB
C.AURANTIBUTYRICUM
C.THERMOBUTYRICUM_SUBGROUP
C. BUTYRICUM
Cyan
High G+C
C.ALGIDICARNIS
Bacteria
Proteo
C.BOTULINUM_SUBGROUP
Bacil-Strep
C.CADAVERIS
Gram +
C.PERFRINGENS
Clostridium
C.BARATI_SUBGROUP 27
1492
16S rDNA
420
469 5 6 7 8
C. perf. resistant
...CGTAAAGCTCTGTCTTTGGGGAAGATAATGACGGTACCCAAGGAGGAAGCCACGGCTAACT... C. perf. str.CPN50
Clostridium sp. AB&J
clone p Wa2
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
Ave Diff =1891
Probe Properties:
25mer exits in 90% of the taxon’s seqs
Internal 21mer exists only in one taxon.
Probes 5 - 8