- #SEQUENCHER COMPARE CHROMATOGRAMS INSTALL#
- #SEQUENCHER COMPARE CHROMATOGRAMS MANUAL#
- #SEQUENCHER COMPARE CHROMATOGRAMS VERIFICATION#
- #SEQUENCHER COMPARE CHROMATOGRAMS SOFTWARE#
Normalize tracesĬomparison of the test to the reference sequence is performed by subtraction of trace values, so it is necessary to normalize the trace values so that a sequence run with strong signal can be meaningfully compared to one with weaker signal. In tests, we established that a filter value of as low as five resulted in the removal most blank terminal sequence, while a value of as high as 500 still retained virtually all genuine sequence we therefore used 50 as an appropriate intermediate value. Blank sequence at the beginning and end of each chromatogram (resulting for example from sequencing of a PCR product, when the trace continues past the end of the template) are removed by deleting the terminal values from the traces where all channel values are less than 50. A, C, G and T) are stored as individual arrays within the chromatogram object. The sequence traces for each channel (i.e. These are uploaded through a web form and the sequence traces and other relevant data extracted using the Perl ABI.pm module. Two ABI sequence chromatograms, one a reference and the other the test sequence, are the only user-supplied data. SeqDoC (Sequence Difference of Chromatograms) is freely accessible, very easy to use, and highlights differences characteristic of single base changes, including heterozygous SNPs and insertions and deletions. 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.
#SEQUENCHER COMPARE CHROMATOGRAMS MANUAL#
This manual approach is affected by variations in sequence quality and incorrect base calling, and may also miss heterozygous bases if, for example, the wild-type peak is higher that the additional peak. Consequently, many small-scale projects may rely solely on manual analysis, for example simply carrying out a direct text comparison of the processed sequence to a known reference. However these are sophisticated and multifunctional programs, and can prove overly complex for simple sequence comparisons.
#SEQUENCHER COMPARE CHROMATOGRAMS SOFTWARE#
In some cases, software such as the Staden package or Sequencher may provide a suitable solution. Identification of point mutations is of equal importance to many researchers, for roles as diverse as identifying specific alterations caused by random mutagenesis screens to validation of the fidelity of sequences amplified by PCR.įor labs studying SNPs or point mutations, identification of these can be a time-consuming and error-prone process, particularly if novel changes are being investigated. The identification and characterisation of naturally-occurring single nucleotide polymorphisms (SNPs) underlies a vast body of work on genetically-linked disorders, diagnosis and risk prediction as well as being important in genomic mapping and population genetics. The ability to identify single nucleotide changes in DNA is a fundamental requirement in many fields of biological research. Differences are highlighted, not interpreted, allowing the investigator to make the ultimate decision on the nature of the change.
#SEQUENCHER COMPARE CHROMATOGRAMS VERIFICATION#
SeqDoC is ideal for small-scale SNP identification, for identification of changes in random mutagenesis screens, and for verification of PCR amplification fidelity. SeqDoC successfully highlights nucleotide changes missed by the Staden package 'tracediff' program. Homozygous and heterozygous substitutions and insertion/deletion events are all readily identified. The use of direct comparison of the sequence chromatograms means that artefacts introduced by automatic base-calling software are avoided. It automatically aligns sequences, and produces straightforward graphical output. SeqDoC produces a subtracted trace showing differences between a reference and test chromatogram, and is optimised to emphasise those characteristic of single base changes.
#SEQUENCHER COMPARE CHROMATOGRAMS INSTALL#
This allows the rapid identification of single nucleotide polymorphisms (SNPs) and point mutations without the need to install or learn more complicated analysis software. This paper describes SeqDoC, a simple, web-based tool to carry out direct comparison of ABI sequence chromatograms.