A tale of two templates: Automatically resolving double traces has many applications, including efficient PCR-based elucidation of alternative splices

Abstract

Trace Recalling is a novel method for deconvoluting double traces that result from simultaneously sequencing two DNA templates. Trace Recalling identifies up to two bases at each position of such a trace. The resulting ambiguity sequence is aligned to the genome, identifying one template sequence. A second template sequence is then inferred from this alignment. This technique makes possible many exciting biological applications. Here we present two such applications, alternate splice finding and elucidation of multiple insertion sites in a random insertional mutagenesis library. Our results demonstrate that RT–PCR followed by Trace Recalling is a more efficient and cost effective way to find alternate splices than traditional methods. We also present a method for mapping double-insertion events in a random insertional-mutagenesis library.