# Presentation: 224978

## Tools used for MSA

- sequences. Find
- near-neighbors.

**Tools used for MSA**

- Trim-away poor quality
- data from a batch of
- sequences.

- Export records from
- the prokMSA.

- Download database,
- presentations,
- and supplemental data.

- Trim

- Align

- Classify

- Classify a querried sequence within a selected database.

- Distance

- Export

- Download

- Calculate a distance matrix.

## What do I do with my 16S reads?

- http://greengenes.lbl.gov
- Todd Z. DeSantis

## What we’ll cover today

- Mini-Background on 16S rRNA gene
- Do alignment
- Do quality assessment, chimera check
- Do classification
- Alternate technology - PhyloChip

## Submit Alignment Job

- Do it now discuss it latter ...
- Every 3rd person?
- Goto: http://greengenes.lbl.gov/Download/Tutorial/
- _[Get: GG_tut_files.zip](http://greengenes.lbl.gov/Download/Tutorial/GG_tut_files.zip)_
- _Unzip it._
- View UnAlignSeqsMGM.fasta in a text editor.
- Goto: _[http://greengenes.lbl.gov](http://greengenes.lbl.gov/)_ then “Align”
- “Submit”

## The Ribosome

- rDNA

- rRNA (functional molecule)

- LSU

- SSU
- 16S or 18S

- Conserved regions at termini allow mixed community PCR amplification.

## Lab Workflows

- Culturable?
- Extractable?
- Able to ligate?
- Able to yield viable  transformants?
- Clean sequencing reaction?

## Classify

- Xie et al. BMC Biology 2004 2:15

- 16S rRNA aids in taxonomic placement of sequences
- Compare your sequence to others.

## Shall we build our house on sand? 

- Shall we build our house on sand?

## Trace - Contig - Collection

- “gg_id” will refer to one contig, replacing prokMSA_id

**Notes:**
Be sure to click to reveal all the other tables

## Greengenes maintains a high-quality Core Set.

- Start by collecting a large set:
- All matching GOLD (genomesonline.org) organisms
*AND*
- All meeting specific criteria
- ≥ 1300 nt.
- ≤ 2 homopolymers
- ≤ 0.3% ambiguity
- ≤ 30 “small gap intrusions” -  bases not supported by the 2° structure.
- Non-chimeric

## Greengenes maintains a high quality Core Set.

- Then de-replicate at >56% common 7mers (Simrank).
- 4,938 genes represents the known 16S diversity.

- Differences between short sequences have a more pronounced effect on Simrank scores.

- Download it to screen your contigs for 16S content...

## NAST align against the core set.

- Example of NAST (Nearest Alignment Space Termination) compression of a BLAST pair-wise alignment using a 38 character aligned template.  Template and candidate is extended to 40 characters after BLAST gap insertion (A) and retention of original template spacing (B). Nucleotide insertions in the candidate relative to the template which force additional characters to be added in the template are identified at positions  and  (C).  A bi-directional search for the nearest alignment space (hyphen) relative to the insertion terminates at the positions indicated by the black arrows (D).  The leftward search from the  position was shorter in distance compared to the rightward, thus the space left of  ‘GT’ was removed.  The search from the  position encountered the alignment edge on the right, thus the position to the left of ‘AC’ was removed (E). Lastly, the two template-extending spaces are deleted from the template (F).  Notice that sequence data is not added to nor overwritten in the candidate. The NAST removal of two characters from both sequences allowed local misalignments (underlined) while preserving the 38 character format of the global multiple sequence alignment.

- Template: ATAC-----GTA-AC----GTA---C---G-T-AC-GG
- Candidate:CACGTTAAACGTCGTACCCGG

- Template: ATACGT-A-ACGTACGTAC-GG
- Candidate:C-ACGTTAAACGT-CGTACCGG

**(A) pair-wise alignment**

- Template: ATAC-----GT-A-AC----GTA---C---G-T-AC--GG
- Candidate:C-AC-----GTTAAAC----GT----C---G-T-ACCCGG

**(B) Re-introduce template spacing**

-                                               
- Template: ATAC-----GT-A-AC----GTA---C---G-T-AC--GG
- Candidate:C-AC-----GTTAAAC----GT----C---G-T-ACCCGG

**(C) Identify template-extending insertions**

**(E) Gap at nearest search terminus in**
**       ****candidate removed **

**(F) Gap in template removed **

**NAST**

**BLAST**

- Template: ATAC-----GTA-AC----GTA---C---G-T-AC-GG
- Candidate:C-AC----_GT_TAAAC----GT----C---G-T_AC_CCGG

**(D) Search for nearest alignment spaces **
**       ****(hyphens) in candidate**

- retained sequence

- Template sequence

**Curated multiple sequence alignment**

- trimmed

- trimmed

**Candidate sequence**

## NAST Alignment Results

- .xls file useful for an overview
- Walk through columns
- NAST vrs NASTnot
- Purpose of nn and nni
- Clear-cut tree ....

## How chimera’s form

## Chimeras are in the public databases.

- Sequence Source

- Sequence Count

## Find them with the partial-partial tree building approach

- Build MSA
- Divide MSA at all possible break points
- Construct 2 distance matrices for each break test.
- Compare consistency of distances.
- (Wang and Wang, 1997; Hugenholtz , 2003)

**1**

**2**

**3**

**4**

**5**

**3**

**4**

**5**

**2**

**1**

## Setting thresholds to flag chimeras.

## Submit Chimera Check 

- Goto: _[http://greengenes.](http://greengenes.lbl.gov/)__[lbl](http://greengenes.lbl.gov/)__[.](http://greengenes.lbl.gov/)__[gov](http://greengenes.lbl.gov/)_
- Then “More Tools”
- Then “Chimera Check with Bellerophon”
- “Submit”

## Taxonomy in Flux

- Incongruent taxonomic nomenclature even at phylum level.
- Making multiple taxonomic classifications available through Greengenes will aid in standardizing classification, particularly for environmental lineages.
- Greengenes integrates each, allowing a balanced approach to nomenclature of newly discovered organisms.
- EXAMPLE: **Halanaerobium salsugo**
- Hugenholtz=Bacteria; Haloanaerobiales; Haloanaerobiaceae; Haloanaerobium/ Haloincola
- Ludwig=Bacteria; Haloanaerobiales; Haloanaerobiacae; Haloanaerobium
- Pace=Bacteria; Haloanaerobiales; Haloanaerobiaceae; Haloanaerobium/ Haloincola
- RDP=Bacteria; Firmicutes; Clostridia; Halanaerobiales; Halanaerobiaceae; Halanaerobium
- NCBI=Bacteria; Firmicutes; Clostridia; Halanaerobiales; Halanaerobiaceae; Halanaerobium

- Each yellow dot represents a named phyla

- Only a fraction of the phyla are recognized by all five major curators.

## Merging Sequencing/PhyloChip Results

- (We can classify when Chimera results return).

## PhyloChip

- Inexpensive
- Do biological replicates
- Pre-screen samples before doing metagenomics
- Rapid
- Comprehensive
- “1 trillion”

## Project Overview

- Goal
- Create a single microarray capable of detecting and categorizing the bacteria in a complex sample.
- Approach
- GeneChip targeted at 16S rDNA sequence variations to distinguish taxa.

## General Protocol

**Air**
**Soil**
**Feces**
**Blood**
**Water**

- rRNA

- gDNA

- Universal 16S rDNA PCR

- Contains probes adhered to glass surface in grid pattern.

## Bacterial species are grouped into OTUs (operational taxonomic units) and detected through a set of probes bound to the chips.  This allows simultaneous detection of multiple known or suspected pathogens as well.

## Hybridization

- Notice all NA is labeled (florescence)
- Non-binding NA is washed away
- If surface “glows”, then target was captured by probe.

- http://www.affymetrix.com

## Millions of copies per feature

## Coordinates of fluorescence determines test results.

## Slide 28

## 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**

## Is  Cyanobacteria OTU 5157 Present?

- Chip says “YES”

**Is  Cyanobacteria OTU 5157 Present?**

- Clone library says “NO”

- PCR with OTU Specific Primers says “YES”

- Sequencing and BLAST of PCR says “YES”

- Confirmation

## Sequence and PhyloChip Integration

## Be sensitive

- Populations with >108 copies in the hybridization are detected

| Total mass | Sensitivity threshold |
| --- | --- |
| 200ng | 0.080% |
| 500ng | 0.030% |
| 1000ng | 0.016% |
| 1500ng | 0.010% |
| 2000ng | 0.008% |

- If I apply this much sample...

- ...then I can detect minority populations down to...

## Quantitative Community Tracking

## Week 1

- Week 2

## Slide 35

## Slide 36

| Family | Class | Peak Duration (sec) |  |
| --- | --- | --- | --- |
| Phaeophyceae (phylum) | Stramenopiles (no rank) | 5 |  |
| Basidiomycota (phylum) | Fungi (kingdom) | 45 |  |
| Family_1.1 | Cyanobacteria | 450 |  |
| Ascomycota (phylum) | Fungi (kingdom) | 450 |  |
| Vibrionaceae | Gammaproteobacteria | 450 |  |
| Flavobacteriaceae | Flavobacteria | 450 |  |
| Clostridiaceae | Clostridia | 45 |  |
| Methylocystaceae | Alphaproteobacteria | 45 |  |
| Acetobacteraceae | Alphaproteobacteria | 45 |  n.s. |
| Rhodospirillaceae | Alphaproteobacteria | 45 |  n.s. |
| Lactobacillaceae | Bacilli | 45 |  |
| Staphylococcaceae | Bacilli | 450 |  |
| Family_3.1 | Cyanobacteria | 450 |  |
| Family_2.2 | Cyanobacteria | 450 |  |
| Mycoplasmataceae | Mollicutes | 5 |  |
| Crenotrichaceae | Sphingobacteria | 5 |  |
| Xanthomonadaceae | Gammaproteobacteria | 0 |  n.s. |
| Burkholderiaceae | Betaproteobacteria | 0 |  |
| Sphingomonadaceae | Alphaproteobacteria | 0 |  |
| Sphingobacteriaceae | Sphingobacteria | 0 |  |
| Acholeplasmataceae | Mollicutes | 45 |  |

## Hierarchical Probes

- Bacteria; Bacteroidetes; BSA2B-18_SP; Unclassified; Unclassified; Unclassified

- Subfamily: sfA
- OTU Count: 2
- Gene Count: 4
- Derep Gene Count: 4

- OTU: 11456
- Gene Count: 3
- Derep Gene Count: 3
- 9 gPM (90% lsp)

- OTU: 11540
- Gene Count: 1
- Derep Gene Count: 1
- 11 gPM (90% lsp)

## Hierarchical Probes

- Bacteria; Chloroflexi; Bljii12_SP; EB1097_CL; AKYG885_OR; Unclassified

- Subfamily: sfA
- OTU Count: 3
- Gene Count: 10
- Derep Gene Count: 10

- OTU: 28140
- Gene Count: 7
- Derep Gene Count: 7
- 2 gPM (90% lsp)

- OTU: 28265
- Gene Count: 1
- Derep Gene Count: 1
- 11 gPM (90% lsp)

- OTU: 28425
- Gene Count: 2
- Derep Gene Count: 2
- 11 gPM (90% lsp)

## PhyloTrac

## Slide 40