# Presentation: 156789

## Detection of chimeric sequences from PCR artefacts

- Thomas Huber _[huber@maths.uq.edu.au](mailto:huber@maths.uq.edu.au)_Computational Biology andBioinformatics EnvironmentComBinE Departments of Biochemistry & Mathematics The University of Queensland

## What are PCR-generated chimeric sequence?

- Prematurely terminated amplicon
- Re-annealing with foreign DNA
- Copied to completion in following PCR cycle
- Artificial sequence from 2 parent sequences

- From: http://www.gnis-pedagogie.org

## Are chimeric sequence a problem?

- Culture independent surveys of microbial communities
- Chimeric sequences suggest non-existing organisms
- 0.5-5% of all sequences are PCR artefacts
- Why bother with such a small artefact?
- Signal vs Noise
- 100 times repetition of same survey (5% chimeras): ratio of existing:non-existing organisms = 1:5

## Detection of chimeras:1. Alignment to reference sequences

- Each target sequence in turn
- Align to ref. sequences
- if alignment to a single sequence gives better match then alignment to two sequences:
-  No chimera
- else:
-  Chimera !!
- (Cole *et a*l., 2003; Komatsoulis and Waterman, 1997, ...)

## Problems

- Database contamination
- More and more chimeras accumulate
- Database coverage
- Parent sequences are not necessarily in database

## 2. Partial tree building approach

- Align sequence to existing sequences (build MSA)
- Divide MSA at postulated conversion point
- Construct 2 trees
- Compare consistency of phylogeny
- (Wang and Wang, 1997; Hugenholtz , 2003)

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## 3. Bellerophon approach

- Just like “partial tree building”, but:
- MSA from PCR library
- More likely to contain parent sequence
- No trees are actually built
- All possible conversion points are tested

## How Bellerophon works

- Compute MSA
- for each conversion point:
- 2 windows left/right
- Calculate all “distances” between sequence
- Instead of comparing trees, compare distance matrices

## How Bellerophon works (cont.)

- Chimeric sequence will result in large dme
- Chimera detection:
- Exclude sequence
- Observe change of dme

## How Bellerophon works (cont.)

- Chimeric sequence will result in large dme
- Chimera detection:
- Exclude sequence
- Observe change of dme

- Expensive to calculate (O(n3))
- Speedy way

## Bellerophon user interface

## Example output

**Title line**

## Example output

**Title line**

**Job parameter**

## Example output

**Title line**

**Job parameter**

**!! Advice !!**

**Chimera output**

## Example output

**Title line**

**Job parameter**

**!! Advice !!**

**Chimera output**

**Preference score (only relative)**

**Conversion points**

**Sequence identities across windows**

**IDs of chimera and parents**

## Server usage

## Who uses Bellerophon?

## What Bellerophon does/does not do!

- Bellerophon does not determine chimeric sequences !!
- It merely indicates putative chimeras
- You _must_ confirm them !

## Current developments

- Bellerophon 2
- For large PCR libraries (or single sequences)
- A smaller library of related sequences is selected for each target sequence
- Cost reduction from O(n3) to  something more tractable
- Cleaning up sequence databases
- Web services
- Large scale data statistics on chimeras

## Bellerophon web services

- Sporadic user (web page interface)
- Interactive / manual use
- Easy to understand, convenient to use
- Large scale users have different needs
- E.g. JGI’s microbial ecology pipeline
- Easy to implement/use interface that allows automatic submission and processing of data
-  Web services
- Standardised protocol (SOAP, WSDL)
- Remote service calls from own scripts and programs
- Not a mirror. All Bellerophon services are maintained in Brisbane

## Large scale data statistics on chimeras

- How much chimeras to expect in a PCR library
- Differences in phyla?
- Is recombination in 16S rRNA a random event?
- Structural bias?