OHSU

Bioinformatics

Free and/or OpenSource Tools

Trouble-shooting automated, fluorescent DNA sequencing data

A handy trouble-shooting guide can be downloaded here. An on-line calculator for DNA unit conversions is available at BioMath.  Please contact theCorelab director for additional help. We have many years of experience and can help with additional data analysis and automation needs as well.

 

DNA Sequence Data: Viewing & Editing

Apple OS X
4Peaks            
Linux FinchTV
Windows XP or 2000

Alternative Base-Caller


SNP Discovery

Fluorescent Sanger sequencing with four dye-labeled terminators produces raw data with fluorescence from the correct, incorporated dideoxy-base but some bleed through from the other three dyes as well. The relative amounts can be quit variable depending on the surrounding sequence. So the raw data is "massaged" (peak shape and spacing) and normalized. The amount of normalization depends on the overall sequence quality. So peak heights are not expected to have a uniform height. This precludes using analyzed sequence data for simple quantification. SNPs from PCR products of heterozygous DNA can be detected, but only their approximate relative peak heights are accessible. You can only say that both peaks are above the normalized noise. If you need to quantitate the contribution of more than a single base at a position you probably want to perform fragment analysis with single base extension or plus/minus ligation.

We offer free sequence comparison using the University of Washington suite of sequence analysis tools, including PolyPhred for SNP analysis.

 

DNA Fragment Analysis Data: Viewing & Editing

Windows XP or 2000
Peak Scanner

Multiple Sequence Alignment

Cross-platform
ClustalX   (easy)       
" T-Coffee (some sys-admin skills required)
" MAFFT ()
"

Tools for alignment note: you must edit your data prior to alignment. This process can be automated, but must be done to avoid spurious alignments. Contact the lab for help.

Primer Design using MIT's Primer3

On-line Primer3 tool