Figure 2

Adaptors and adaptor chimeras are a common sources of sequence artifacts. Specific outer adapter sequences, complementary to the grafting sequences on the flow cell are essentially the only requirement for sequencing a DNA library on the Genome Analyzer platform. As different sequencing primers can be used, library design is very flexible and various protocols with partially distinct adapter sequences have been established. The Illumina NlaIII DGE tag protocol illustrated here (a protocol for digital gene expression tag profiling) uses short adapters which are not compatible with paired end sequencing and are added by overhang ligation (A). For this protocol the majority of adapter dimers are removed by a gel excision step after library preparation. However, the protocol may also create adapter chimeras with a length comparable to the targeted library molecules. The resulting chimera sequences also show the sequences required for cluster generation as well as the necessary priming site, causing them to be sequenced together with the real DGE tags. A program like TagDust [29] can be used with the original adapter and primer oligonucleotide sequences to identify such artifacts (B). Shown are the twenty most frequent identified artifacts from one lane with human DGE tags, as well as the oligosequences they might be based on. One of the 20 sequences seems to be a real DGE tag that was incorrectly identified as an artifact.