# Presentation: 380717

## Pick a bug, any bug.

- The design and practical use of rDNA oligonucleotide microarrays to identify microbes in complex samples
- Todd DeSantis, Igor Dubosarskiy (Ceres), Sonya Murray, Gary Andersen
- Environmental Molecular Microbiology Group
- BBRP - LLNL

- Comprehensive Aligned Sequence Construction for Automated Design of Effective Probes (CASCADE-P)

## The worries of an Italian parent

**The worries of an ****Italian**** parent**

- Is he getting enough sleep?

- Does he feel accepted?

- Is there sufficient diversity in his lower G.I. bacterial community?

- Are the straps on his car seat irritating his neck?

- Are there archaeal organisms are in the aerosols he breathes?

- Enzo Salvatore DeSantis

- Jenny DiGiovanni DeSantis

## Every soiled diaper sacrificed to the Diaper Genie is lost data.
But who wants to do all the work?
Culture
anaerobes
non-cultivable
Sequencing 16S rDNA
Need to create, clone, & process hundreds of samples 
Can we create a simple, quantitative, comprehensive microbial test?

- But who wants to do all the work?
- Culture
- anaerobes
- non-cultivable
- Sequencing 16S rDNA
- Need to create, clone, & process hundreds of samples
- Can we create a simple, quantitative, comprehensive microbial test?

## Outline

- Goals
- Experimental approach
- Why create a new 16S rDNA database?
- How do you align >65,000 sequences?
- Organization of sequences into* types*
- Designing probes for each *type*
- Reassessing probe specificity as database grows
- Using 16S GeneChip for quantitative aerosol analysis

## Project Overview

- Create a single GeneChip® capable of detecting and quantifying bacterial and/or archaeal organisms in a complex sample.
- What *is *in a sample, as opposed to what *is* *not* in a sample.
- Approach
- Combinatorial power of  multiple probes for sequence-specific hybridization

## General Protocol

- Sample
- Air
- Soil
- Feces
- Blood

- rRNA

- gDNA

- Universal 16S rDNA PCR

- Random hexamer total gDNA amplification
- Allen Christian

- Contains probes adhered to glass surface in grid pattern.

## 16S rRNA gene (16S rDNA)

- Used to identify and classify organisms by gene sequence variations.
- Variations have been used in design of DNA probes for the detection of:
- taxonomic domains, divisions, groups ...
- specific organisms

## The Ribosome

**The Ribosome**

- rDNA

- rRNA (functional molecule)

- LSU

- SSU
- 16s or 18s

## The Ribosome

**The Ribosome**

- Folded secondary structure
- Essential functional component
- Conserved spans
- structure must be retained for viability
- targeted for universal/group-specific PCR primers and probes
- Variable regions
- spans not fundamental to the folded structure
- receive less pressure from natural selection
- probed for genus and species level discrimination

## Building upon two decades of 16S gene cataloging

- Over 75,000 16S records housed at NCBI.
- Grows every week.
- Don’t need to build a reference library for organism ID.
- FAME
- MS
- Surface Antigen Identification

## What could be amplified?

- Universal 16S PCR primers  complex population of amplicons.
- Must define the targets to consider as the Potential Amplicon Set or **PAS**.
- “Give me a file of all the amplicon sequences possible if we used universal primers upon a sample containing every prokaryote.”

- Variable

- Tom Kusmarski

## Difficulties defining the PAS

- Why Entrez search for “16S” is insufficient:
- non-16S sequences are errantly deposited as 16S
- 16S sequences may be concealed within longer records that cover entire operons or genomes
- anti-sense strands aren't specified as such

## Difficulties defining the PAS

- For each sequence, need to trim away bases that won’t be amplified.
- Problem: Most 16S records are “partial”.
- Can primer pattern matching within sequences allow for proper trimming?
- What does it mean when a primer search fails?
- Primer locus present in record but mutated, or
- Primer locus outside the sequence span deposited

## Difficulties defining the PAS

- Aligned sequences were necessary.
-  A 16S MSA arranged as horizontal rows of characters allows vertical slices to be extracted between columns of primer annealing positions.

## Existing 16S aligned databases

- Under 20,000 sequences among 3 databases.
- Updates occurred annually or worse.
- Others focused on hand-aligning complete sequences.
- Structure predictions
- Phylogeny assessment
- Comprehensive Aligned Sequence Construction for Automated Design of Effective Probes (CASCADE-P)
- need up-to-date records
- need to include partial sequences
- add “inertia” to region considered conserved
- increase the likelihood of detecting a polymorphism
- searched for unwanted cross-hybridizations with a tentative probe

## RDP alignment and tree – a great skeleton

**RDP alignment and tree – a great skeleton**

- Ribosomal Database Project (Michigan State)
- 16S MSA of 16 277 seqs (v8.1)
- trimmed of extra-16S
- top-strand oriented
- 1,541 bases stretched to 4,218 characters
- Each placed within a hierarchical phylogenetic tree

## Sequence pre-processing

- Download ‘16S’ candidates via “ESearch”
- BLAST compare to 16S/18S RDP “standards”.
- Candidates were rejected if
- the longest match length was <300 base pairs
- the highest scoring BLAST subject was eukaryotic
- candidate matched sequences in two or more RDP terminal tree branches equally well
- Phylocode assigned from top HSP

## Sequence pre-processing

## Sequence pre-processing

- Candidate trimmed of extra-16S seq data
-  tRNA genes, intergenic spacer regions, and 23S rDNA
- based on HSP boundries
- If HSP paired opposite strands, candidate was reverse complemented.

## Sequence pre-processing

## Sequence pre-processing

- The "template" was assigned from the top HSP from a second BLAST process
- G=1, E=1.
- Favors longer, but less identical matches.

## Prokaryotic Multiple Sequence Alignment prokMSA

- Essentially, the prokMSA was a merger built serially by aligning each candidate to its closest relative in the RDP tree.
- Two Steps
- Align0
- Publicly available, pair-wise SW aligner
- Candidate expansion
- NAST
- Nearest Alignment Space Termination
- Novel algorithm
- Candidate compression

## NAST

**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**

## December 28th, 2002

- December 28th, 2002

## Operational Taxonomic Units

- We *did not* desire to design probes for each sequence.
- Many sequences are nearly identical.
- Desired 20 probe per sequence:
- 60,000 seqs * ( 20 probes + 20 probes)  =
- 2.4 million probes (not possible, yet)
- We *did* desire to design probes for each *type* of sequence.
- Need to group sequences into *types* amenable to probe design.

## Operational Taxonomic Units

- Avoid groupings based on historical nomenclature.
- Sequence-dependent classification by transitive similarity clustering at 98%.
- Create groupings into Operational Taxonomic Units (OTU).
- Each sequence must be in exactly 1 OTU

**if   x R y   &   y R z  ********   x R z **

## Sequences Clustered

** ****GRAM_POSITIVE_BACTERIA** (2.30)
-   **BACILLUS-LACTO-STREPTOCOC_SUBDIVISION** (2.30.7)
-    **CARNOBACTERIUM_GROUP** (2.30.7.18)
-     **CRN.DIVERGENS_SUBGROUP** (2.30.7.18.2)
-      OTU 2.30.7.18.2.012 (11 sequence records)
-        AF244371 Nostocoida limicola I Ben200
-        AF244372 Nostocoida limicola I Ben201
-        AF244375 Nostocoida limicola I Ben77
-        AF255736 Nostocoida limicola I
-        AF276462 Uncultured bacterium clone RFLP 102E
-        AF394926 Lactosphaera sp. PMagG1
-        AJ296179 Ruminococcus palustris DSM 9172T
-        AJ306612 Trichococcus collinsii 37AN3
-        L76599   Lactosphaera pasteurii ATCC 35945
-        X87150   Lactosphaera pasteurii DSM 2381
-        Y17301   Trichococcus flocculiformis DSM 2094

**Sequences Clustered**

***I******s***** = **** *****I******B****** ********* L****m**** / min (L****a, ****L****b****)**

## Bioinformatics manuscript input 

**Some OTUs contain hundreds of sequences.**
**Example: Many isolates of a human pathogen.**

**Some “species” are found in over 20 OTUs.**

_*Bioinformatics *__manuscript input _
- Sonya Murray
- Peter Agron
- Sadhana Chauhan

## http://greengenes.llnl.gov/16S

**C**omprehensive **A**ligned **S**equence **C**onstruction for **A**utomated **D**esign of **E**ffective **P**robes
- Igor Dubosarskiy
- Java implementations
- Tim Harsch
- RDBMS consultations
- Lisa Corsetti
- Apache module management
- Kevin Melissare
- Graphics

## “My Interest List”

- Able to define a region of the tree that is important to you.
- Your list will be remembered between visits.

## Picking Probes for GeneChip Microarray

- 27

- 1507

**pA**

**1492R**

- Select 20 to 28 probe pairs for each of 8,432 OTUs
- Ideal Perfect Match Probe
- 25mer
- Present in all sequences of the OTU
- Not present outside the OTU
- Unable to X-hybe with seqs in other OTUs
- Ideal Mis-match Control Probe
- Unable to X-hybe within entire PAS

- Slice taken from prokMSA

## Scoring Probe Candidates

- Score is calculated for each potential probe pair.
-  Product of 3 factors:
- Locus Specific Prevalence Factor
- PerfectMatch X-hybe Factor
- The closer the tree distance, the lower the factor
- MisMatch X-hybe Factor
- 3 bases available
- use base which produces highest factor

## Locus Specific Prevalence Scoring

- OTU composed of _**26**_ sequences

## Cross Hybridization

- Central Data Structure:
- $17mer_hash{AGCTATTATAGCTGCAG}{‘2.30.7.12.4.004’} = 1
- $17mer_hash{AGCTATTATAGCTGCAG}{‘2.30.7.12.4.009’} = 1
- $17mer_hash{AGCTATTATAGCTGCAG}{‘2.30.7.12.3.001’} = 1
- ...
- 1.2 Gb
- Allowed rapid lookup of all OTUs containing a particular 17mer

- _Consultations_:
- Mark Wagner
- Tom Slezak
- Tom Kuczmarski
- Mike Mittman, Affymetrix

## OTU-2.30.7.18.2.002

**OTU-2.30.7.18.2.002**

- Sample Probe Rankings

**RANK**

## Combinatorial scoring of “Probe Sets” are able to categorize mixed samples.

*S. aureus *spike

*B. anthracis *spike

- Art Koybayashi – Simulation package

## Combinatorial scoring of “Probe Sets” are able to categorize mixed samples.

- Percent of probe-pairs scored positive for each probe set in the Staphylococcus Group.

- Hybridization results from spike-in experiment done in triplicate.
- Sonya Murray
- Aubree Hubbel

**Combinatorial scoring of “Probe Sets” are able to categorize mixed samples.**

## PAS is a moving target

- Problems
- New 16S sequence data is constantly being deposited to public databases.
- Mismatch Probes can become Perfect Matches.
- Phylogenetic groupings (OTUs) can change.
- New transitive links may be discovered

## Dynamic probe associations

- BlockNumber=1
- NumAtoms=33
- NumCells=66
- CellHeader=X    Y       PROBE   FEAT    QUAL
- Cell1=6         59      TCAAACATTGCGGGCTTCAG    2.30.7.12.004
- Cell2=6         58      TCAAACATTGTGGGCTTCAG    2.30.7.12.004
- Cell3=171       202     AAACATTGTGGGCTTCAGCC    2.30.7.12.004
- Cell4=171       203     AAACATTGTGTGCTTCAGCC    2.30.7.12.004
- Cell5=197       163     CATTGTGGGCATCAGCCACC    2.30.7.12.004
- Cell6=197       162     CATTGTGGGCTTCAGCCACC    2.30.7.12.004
- Cell7=151       175     ATTGTGGGCTGCAGCCACCC    2.30.7.12.004
- Cell8=151       174     ATTGTGGGCTTCAGCCACCC    2.30.7.12.004
- Cell9=228       2       TTGTGGGCTTCAGCCACCCC    2.30.7.12.004
- Cell10=228      3       TTGTGGGCTTTAGCCACCCC    2.30.7.12.004
- Cell11=139      22      TGTGGGCTTCAGCCACCCCA    2.30.7.12.004
- Cell12=139      23      TGTGGGCTTCGGCCACCCCA    2.30.7.12.004
- Cell13=94       76      GGGCTTCAGCCACCCCATTG    2.30.7.12.004
- Cell14=94       77      GGGCTTCAGCTACCCCATTG    2.30.7.12.004
- Cell15=111      118     CTTCAGCCACCCCATTGGAA    2.30.7.12.004
- ...
- ...
- ...
- ...
- ...
- ...

_**Dynamic**_** probe associations**

- Ability to “re-map” chip to up-to-date 16S data
- Many of the existing probes on the physical array are complementary to “new” sequences.
- Probes originally deemed MM can become PM to some organisms.
- mySQL database used for association maintenance

- original (static)

- dynamic

## Finding groupings

**Finding groupings**

| seq | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| B |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| C |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| D |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| E |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| F |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| G |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| H |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| I |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| J |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| K |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| L |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| M |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| N |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| O |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

- sequences

- probes

- Consider A – O to be 16S sequences.
- Consider 1 – 24 to be probes already embedded on the chip.
- First, associate all available probes with all available sequences.
- Let probe similarities drive sequence groupings.

## Finding groupings

| seq | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| B |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| C |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| D |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| E |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| F |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| G |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| H |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| I |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| J |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| K |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| L |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| M |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| N |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| O |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

- Consider A – O to be 16S sequences.
- Consider 1 – 24 to be probes already embedded on the chip.
- First, associate all available probes with all available sequences.
- Let probe similarities drive sequence groupings.

## Finding groupings

| seq | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| B |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| C |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| D |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| E |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| F |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| G |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| H |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| I |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| J |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| K |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| L |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| M |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| N |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| O |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

- Consider A – O to be 16S sequences.
- Consider 1 – 24 to be probes already embedded on the chip.
- First, associate all available probes with all available sequences.
- Let probe similarities drive sequence groupings.

## Finding groupings

| seq | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| B |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| C |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| D |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| E |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| F |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| G |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| H |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| I |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| J |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| K |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| L |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| M |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| N |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| O |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

- Consider A – O to be 16S sequences.
- Consider 1 – 24 to be probes already embedded on the chip.
- First, associate all available probes with all available sequences.
- Let probe similarities drive sequence groupings.

## Finding groupings

| seq | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| B |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| C |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| D |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| E |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| F |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| G |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| H |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| I |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| J |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| K |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| L |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| M |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| N |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| O |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

- Consider A – O to be 16S sequences.
- Consider 1 – 24 to be probes already embedded on the chip.
- First, associate all available probes with all available sequences.
- Let probe similarities drive sequence groupings.

## Progressive Cyclical Grouping

**DEFINE** *uGBpp* as the number of useful probe pairs which globally differentiate a cluster from all other sequences.
**FOR** *uGBpp**lock* (11 .. 4) DO
**	****FOR** *uPWpp**sep* (1 .. 10) DO
- 		Determine *uGBpp**clust** *for each cluster.
- 		Lock all clusters where *uGBpp**clust *≥ *uGBpp**lock** *.
- 		Pair-wise (PW) compare each non-locked cluster (*clustA, clustB*)
*	**	**uPWpp**clustA** = *number of useful probe pairs which PW differentiate *clustA* from *clustB *
*	**	**uPWpp**clustB** = *number of useful probe pairs which PW differentiate *clustB* from *clustA*
- 		Merge sequences of *clustA* and *clustB* into one cluster unless *uPWpp**clustA** *≥ *uPWpp**sep *AND *uPWpp**clustB** *≥ *uPWpp**sep*
**         ****END FOR**
- 	Separate all sequences in non-locked clusters so that each sequence is the sole element of its own cluster.
**END FOR**

## Quantitative Analysis

- Could the concentration of each amplicon in a sample be measured by fluorescence intensity?
- Experimental setup for 20 point calibration:

| Experiment
 | Lc.oenos | Fer.nod | Sap.grand | M.neuro |  | H20 | 16S amplicons* |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 5 | 13 | 31 | 74 |  | No | Yes |
| 2 | 13 | 31 | 74 | 143 |  | No | Yes |
| 3 | 31 | 74 | 143 | 5 |  | No | Yes |
| 4 | 74 | 143 | 5 | 13 |  | No | Yes |
| 5 | 143 | 5 | 13 | 31 |  | No | Yes |
| 6 | 0 | 0 | 0 | 0 |  | Yes | Yes |

- * 18uL of products from 30 cycle universal 16S PCR of gDNA extracted from U.K. air sample.

- SPIKE CONCENTRATION  (pM in Hybridization Solution)

- Sonya Murray
- Carol Stone

## Quantitative Analysis

- Log2 transformed
- Linear Least Squares Regression
- Pearson’s corr coeff was significant (df=18)
- 95% confidence intervals calculated according to: National Measurement System _V_alid _A_nalytical _M_easurement Programme (VAM)

## Quantitative Analysis

- Environmental community is measured with confidence intervals.

## Summary

- prokMSA contains 65,000 aligned sequences and growing (largest collection).
- Over 8,000 distinct OTUs have been found.
- Global probe-picking was completed.
- “DOE 16S” GeneChip® was manufactured.
- Ability to correctly categorize spike-ins is being validated.
- Detected amplicons can be quantified.

## Acknowledgements

- Gary Andersen – Group Leader (The Tangent Terminator)
- Carol Stone – Sample collection, hybridization (DSLT)
- Aubree Hubbel – Spike synthesis
- Sonya Murray - Hybridizations
- Peter Agron – ms advise
- Sadhana Chauhan – ms advice
- Mike Mittman – probe selection constraints (Affymetrix)
- Art Koybayashi – Hyb simulation package, ms advice
- Tom Kusmarski  - PAS, algorithm optimization
- Tom Slezak - algorithm optimization
- Mark Wagner - algorithm optimization
- Igor Dubosarskiy – Java, web front-end (Ceres)
- Tim Harsch - RDBMS
- Lisa Corsetti – Apache administration
- Allen Christensen – genomic sample amplification
- This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory, under contract no. W-7405-Eng-48.

## Slide 51