Extraction of SAGE tags from sequences/chromatographs and error correction methods

Motivation:

Sequencing errors may bias the gene expression measurements made by SAGE. They may introduce non-existent tags at low abundance and decrease the real abundance of other tags. These effects are increased in the longer tags generated in LongSAGE libraries. Current sequencing technology generates quite accurate estimates of sequencing error rates. Here we make use of the sequence neighborhood of SAGE tags and error estimates from the base-calling software to correct for such errors.

Results:

We introduce a statistical model for the propagation of sequencing errors in SAGE and suggest an Expectation-Maximization (EM) algorithm to correct for them given observed sequences in a library and base-calling error estimates. We tested our method using simulated and experimental SAGE libraries. When comparing SAGE libraries, we found that sequencing errors can introduce considerable bias. High abundance tags may be falsely called as significantly differentially expressed, especially when comparing libraries with different levels of sequencing errors and/or of different size. Truly differentially expressed tags have decreased significance as "true"-tag counts are generally underestimated. This may alter if tags near the threshold of differential expression are called significant. Moreover, the number of different transcripts present in a library is overestimated as false tags are introduced at low abundance. Our correction method adjusts the tag counts to be closer to the true counts and is able to partly correct for biases introduced by sequencing errors.

Availability:

Use methods online
An implementation using R is distributed as an R package. Build for 2.0: sagenhaft_0.3.tar.gz, sagenhaft_0.3.zip. In R install with install.packages("sagenhaft", contriburl="http://tagcalling.mbgproject.org/").

Cite:

Statistical modeling of sequencing errors in SAGE libraries
Beissbarth T, Hyde L, Smyth GK, Job C, Boon WM, Tan SS, Scott HS, Speed TP
Bioinformatics; 7.2004; 20(ISMB Supplement 1), I31-I39.

Contact:

Tim Beissbarth - beissbarth@wehi.edu.au
Last modified: 09.09.2004

Bioinformatics

Melbourne Brain Genome Project

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