# Presentation: 529827

## greengenes.lbl.gov 16S rRNA gene database and workbench compatible with ARB 

- <date/time>

- _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

## 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

**Source: **_[http://www.ncbi.nlm.nih.gov/](http://www.ncbi.nlm.nih.gov/)_**  **
**‘****16S NOT 1.16S NOTmitochondr* NOT 18S’**

## 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%) |
|  |  |

- greengenes.lbl.govVerify ‘16S-ness’

## NAST alignstep 1: find template

- 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 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

- 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 alignstep 1: find template

- 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 alignstep 2: gap removal

**Preserves global MSA positions(columns) by allowing local misalignments.**
**DEFINE**
*S**t* = post-Align0 template sequence.
*S**c* = post-Align0 candidate sequence.
*H**t* = alignment space (hyphen) inserted into *S**t* by Align0.
*H**c* = alignment space (hyphen) inserted into *S**c* by Align0.
**WHILE** (*S**t* contains one or more *H**t*) **DO**
- 	*L**Ht* = character index of distal 5' *H**t* within *S**t*
- 	*L**5'* = character index of *H**c* within *S**c* which is 5' proximal to *H**t*
- 	*L**3'* = character index of *H**c* within *S**c* which is 3' proximal to *H**t*
- 	**IF** ((*L**Ht* – *L**5'*) > (*L**3'* – *L**Ht*))  Delete *H**c* found at *L**3'*
*	**     ***ELSE**       Delete *H**c* found at *L**5'*
- 	Delete template gap character.
**END WHILE**
**Result: Largest MSA of full-length (>1250  nt) 16S rDNA genes.**

## greengenes.lbl.govName generator

- <date/time>

- 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 **isolate**

- yes

- no

- 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

- no

- yes

- Does a source>isolate field  exist?

- yes

- no

- Record is from **undecided**

- “Gs no”

- yes

- no

- “Isolate tag no”

- “Isolate tag yes”

- Gs result?

-  if Gs

- yes

- no

- Strain tag is present

- Record is from a **isolate_str**

- greengenes.lbl.govName 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/](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 Taxonomy

**JGI taxonomy organized in ARB using maximum parsimony tree insertions.**
**Example: **_[http://greengenes.lbl.gov/](http://greengenes.lbl.gov/cgi-bin/User/show_one_record_v2.pl?prokMSA_id=82172)__[cgi](http://greengenes.lbl.gov/cgi-bin/User/show_one_record_v2.pl?prokMSA_id=82172)__[-bin/User/show_one_record_v2.pl?prokMSA_id=82172](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](http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide&dbFrom=nucleotide&cmd=search&term=28971862&doptcmdl=GenBank&tool=prokMSA)_, GenBank GI: 28971862, RDP_id: _[S000122947](http://rdp.cme.msu.edu/hierarchy/detail.jsp?seqid=S000122947)_, NCBI_tax_id: _[203524](http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=203524)_, Study_id: _[21358](http://greengenes.lbl.gov/cgi-bin/User/show_one_study.pl?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 Taxonomy

- <date/time>

- 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)

## Probe Design

-  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**

## Locus Specific Prevalence Scoring

- Example: proteobacteria OTU composed of _**26**_ sequences

## 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.

## Overview of Sample Preparation

- 18 μ

- 18 μ

**A**
**C**
**G**
**G**
**T**
**C**
**G**
**A**

**A**
**C**
**G**
**G**
**T**
**C**
**G**
**A**

**A**
**C**
**G**
**G**
**T**
**C**
**G**
**A**

**A**
**C**
**G**
**G**
**T**
**C**
**G**
**A**

**A**
**C**
**G**
**G**
**T**
**C**
**G**
**A**

**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

- Scores for each of 9000 OTUS

## 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

## 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 | 50.9 |
| Fervidobacterium nodosum | 58.2 | 53.8 |
| Caulobacter vibrioides | 56.4 | 58.5 |

## Array is quantitative

**~10****7**** 16S gene copies**

**~10****11**** 16S gene copies**

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

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

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

| Representative organism | Phylocode | Group | Corrected 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 |

- (a) Array quantitation

- (b) qPCR quantitation

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

## Real-time quantitative PCR confirmation – Urban Aerosol

- Array hybridization signal correlates significantly
- with 16S copies in environmental aerosol DNA extract
*Pseudomonas oleovorans* example

## FEMS Letters - pseudoshift

| 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 |  |

## 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

## C. perfringens probe set identified in 

**...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 seqs
- Internal 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)**