Abstract
BackgroundThis paper describes SeqDoC, a simple, web-based tool to carry out direct comparison of ABI sequence chromatograms. This allows the rapid identification of single nucleotide polymorphisms (SNPs) and point mutations without the need to install or learn more complicated analysis software.ResultsSeqDoC produces a subtracted trace showing differences between a reference and test chromatogram, and is optimised to emphasise those characteristic of single base changes. It automatically aligns sequences, and produces straightforward graphical output. The use of direct comparison of the sequence chromatograms means that artefacts introduced by automatic base-calling software are avoided. Homozygous and heterozygous substitutions and insertion/deletion events are all readily identified. SeqDoC successfully highlights nucleotide changes missed by the Staden package 'tracediff' program.ConclusionSeqDoC is ideal for small-scale SNP identification, for identification of changes in random mutagenesis screens, and for verification of PCR amplification fidelity. Differences are highlighted, not interpreted, allowing the investigator to make the ultimate decision on the nature of the change.
Highlights
This paper describes SeqDoC, a simple, web-based tool to carry out direct comparison of ABI sequence chromatograms
Identification of point mutations is of equal importance to many researchers, for roles as diverse as identifying specific alterations caused by random mutagenesis screens [9,10] to validation of the fidelity of sequences amplified by PCR
BMC Bioinformatics 2005, 6:133 http://www.biomedcentral.com/1471-2105/6/133 carrying out a direct text comparison of the processed sequence on one side, while still using 500 on the other sequence to a known reference. This manual approach is affected by variations in sequence quality and incorrect base calling, and may miss heterozygous bases if, for example, the wild-type peak is higher that the additional peak. To address these issues and to provide a simple and efficient way to accurately identify sequence changes, we have developed a web-based application which compares DNA sequence chromatograms directly
Summary
SeqDoC produces a subtracted trace showing differences between a reference and test chromatogram, and is optimised to emphasise those characteristic of single base changes. It automatically aligns sequences, and produces straightforward graphical output. The use of direct comparison of the sequence chromatograms means that artefacts introduced by automatic basecalling software are avoided. Homozygous and heterozygous substitutions and insertion/deletion events are all readily identified. SeqDoC successfully highlights nucleotide changes missed by the Staden package 'tracediff' program
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