%0 Journal Article
%T A direct comparison of the KB£¿ Basecaller and phred for identifying the bases from DNA sequencing using chain termination chemistry
%A Richard W Hyman
%A Hui Jiang
%A Marilyn Fukushima
%A Ronald W Davis
%J BMC Research Notes
%D 2010
%I BioMed Central
%R 10.1186/1756-0500-3-257
%X The high quality sequence segment of reads derived from the KB£¿ Basecaller were, on average, 30-to-50 bases longer than reads derived from phred. However, microbe identification appeared to have been unaffected by the change in software.We have demonstrated a modest, but statistically significant, superiority in high quality read length of the KB£¿ Basecaller compared to phred. We found no statistically significant difference between the numbers of microbial species identified from the sequence data.DNA sequencing by DNA polymerase chain termination was introduced by Sanger et al. [1] in 1977. In this technology, sequence is determined from the lengths of the terminated DNA chains. Electrophoresis is employed to separate the chains based upon length. A different fluorescent dye is covalently attached to each of the four dideoxy chain terminators. The presence of the dyes significantly affects the electrophoretic mobility of the chains. Therefore, sophisticated software must be employed to deconvolute the fluorescent signals into bases.For some years, the suite of software of choice for DNA sequencing was introduced by Green and associates in 1998: phred for calling the bases in sequence reads, phrap for assembling the reads into contigs, and consed for displaying the contigs for editing [2-4]. Relatively recently, the manufacturer of the sequencing equipment, Applied Biosystems (ABI, Foster City, CA), introduced its own base calling software, the KB£¿ Basecaller, to replace phred http:/ / www3.appliedbiosystems.com/ cms/ groups/ mcb_marketing/ documents/ generaldocuments/ cms_040412.pdf webcite.In our published study [5], we identified the microbes in the healthy adult human vagina by PCR amplifying the 16S ribosomal RNA genes, sequencing the genes with dideoxy chemistry, and identifying the microbes by comparison of the sequence to the data in the Ribosomal Database Project (RDP) [6]. We were concerned that the change in base-calling software would change the microbe
%U http://www.biomedcentral.com/1756-0500/3/257