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Multicenter Study
. 2006 Apr;17(2):138-44.

Evaluation of methods for sequence analysis of highly repetitive DNA templates

John W Hawes  1 , Kevin L Knudtson, Helaman Escobar, George S Grills, Timothy C Hunter, Emily Jackson-Machelski, Heather Lin, David S Needleman, Rashmi Pershad, Glenis J Wiebe
Affiliations
Multicenter Study

Evaluation of methods for sequence analysis of highly repetitive DNA templates

John W Hawes et al. J Biomol Tech. 2006 Apr.

Abstract

The DNA Sequencing Research Group (DSRG) of the ABRF conducted a study to assess the ability of DNA sequencing core facilities to successfully sequence a set of well-defined templates containing difficult repeats. The aim of this study was to determine whether repetitive templates could be sequenced accurately by using equipment and chemistries currently utilized in participating sequencing laboratories. The effects of primer and template concentrations, sequencing chemistries, additives, and instrument formats on the ability to successfully sequence repeat elements were examined. The first part of this study was an analysis of the results of 361 chromatograms from participants representing 40 different laboratories who attempted to sequence a panel of difficult-to-sequence templates using their best in-house protocols. The second part of this study was a smaller multi-laboratory evaluation of a single robust protocol with the same panel of templates. This study provides a measure of the potential success of different approaches to sequencing across homopolymer tracts and repetitive elements.

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Figures

FIGURE 1
FIGURE 1
Illustration of the repetitive elements contained in the templates A, B, and C. The number of bases downstream of the M13 primer binding site is shown on top. Template A contained a 20-fold repeat of TTTC followed by a 12-fold repeat of CCCT followed immediately by a 26 base-pair repeat of CT. These repetitive elements began at approximately 250 base pairs from the M13 primer binding site. Template B contained an initial A-rich sequence containing two repeats of AAAATTCT at position 165 after the M13 primer binding site. This short repetitive element was followed by a 7-fold repeat of AAGG, a 30-fold repeat of AGGG, and an approximately 30-base-pair repeat of AG. Template C represented a greater level of diversity in the repetitive elements. This template contained a 44-base-pair repeat of GT starting at position 156 after the M13 primer binding site, followed immediately by a 32-base-pair repeat of AG and a 10-fold repeat of CCGA.
FIGURE 2
FIGURE 2
The effect of the amount of primer on sequence quality. A:Results displayed as the exact amount of primer added. B: Primer amounts adjusted for the reaction volume. The bars represent the average and standard errors of the q20 scores for each template (A, B, and C) for the amount of primer used in the sequencing reaction.
FIGURE 3
FIGURE 3
The effect of the amount of template on sequence quality. A:Results displayed as the exact amount of template added. B: Template amounts adjusted for the reaction volume. The bars represent the average and standard errors of the q20 scores for each template (A, B, and C) for the amount of template used in the sequencing reaction.
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