Info

fluorescence information via both color and lifetime is attractive for high multiplexing opportunities in DNA sequencing.

A two-color/two-lifetime sequencing run was carried out as an initial test of the color and time-resolve hybrid system approach (Zhu et al., 2003). Two dye pairs, IRD700 and AlexaFluor680, were processed in a 710-nm color channel, and IRD800 and near-IR-Br dye (see Table 7.2) [93] were used in an 810-nm color channel. Each pair of dyes showed minimal differences in their excitation and emission profiles, but possessed distinguishable lifetimes in the sequencing matrix used for electrophoretic sorting of the DNA. The decay profiles for all four dyes as well as the instrument response function in each color channel are shown in Figure 7.8. Four single-base tracts were run prior to the sequencing experiment to determine the lifetime value of each dye label under typical electrophoresis conditions. The average lifetime values, calculated using the MLE algorithm, for each dye label were 913 ± 9 ps, 1493 ± 11 ps, 454 ± 7 ps, and 744 ± 16 ps, for IRD700, Alexa680, IRD800, and NIR-Br, respectively. Based on the predetermined lifetime values, an automatic peak recognition algorithm was applied to assist the identification of the terminal bases. The raw data were subjected to mobility shift corrections but without going through any other data

Figure 7.8. Instrument response function and fluorescence decay profiles for a two-color/two lifetime hybrid instrument. (a) Fluorescence decay profiles for IRD700-labeled G- and AlexaFluor680-labeled T fragments as well as the instrument response function for the 710-nm color channel. (b) Fluorescence decay profiles for IRD800-labeled C- and NIR-Br-labeled A-sequencing fragments and the instrument response function generated in the 810-nm color channel. Individual dye-labeled DNA ladders were analyzed using CGE with POP6 as the sieving matrix. Decays were constructed by integrating photocounts over 5 pixels (integration time of 5 s) centered on individual electrophoretic peaks from each sequencing trace. The instrument response functions were accumulated over 5 pixels from the gel track prior to migration of the DNA fragments into the detection volume (Zhu et al., 2003). See insert for color representation of this figure.

Figure 7.8. Instrument response function and fluorescence decay profiles for a two-color/two lifetime hybrid instrument. (a) Fluorescence decay profiles for IRD700-labeled G- and AlexaFluor680-labeled T fragments as well as the instrument response function for the 710-nm color channel. (b) Fluorescence decay profiles for IRD800-labeled C- and NIR-Br-labeled A-sequencing fragments and the instrument response function generated in the 810-nm color channel. Individual dye-labeled DNA ladders were analyzed using CGE with POP6 as the sieving matrix. Decays were constructed by integrating photocounts over 5 pixels (integration time of 5 s) centered on individual electrophoretic peaks from each sequencing trace. The instrument response functions were accumulated over 5 pixels from the gel track prior to migration of the DNA fragments into the detection volume (Zhu et al., 2003). See insert for color representation of this figure.

manipulations that are normally involved in many automated sequencing machines, such as removal of cross-talk, baseline adjustment, or signal normalization. The calling accuracy was 95.1% over a read length of 650 base pairs, with the majority of errors occurring at late times within the electrophoresis trace due to poor separation efficiency at the end of the run.

The same strategy (two-color/two-lifetime) was also tested on a microchip electrophoresis format (Zhu et al., 2004). The sequencing samples were required to be purified and pre-concentrated prior to electrophoretic sorting to improve the precision of the lifetime determinations due to the low loading levels associated with the microchip format. The fluorescence lifetimes of all dyes were determined with favorable precisions on the microchips.

7.4.2. Potential Applications

The primary motivation for coupling time-resolved measurements with color discrimination is to increase the fluorescence multiplexing capability to increase the information content obtainable in a single gel lane, thereby increasing the data throughput. With appropriate dye sets, a two-color, four-lifetime sequencing strategy could be envisioned to allow the identification of eight unique reporters. This scheme could, for example, allow simultaneous forward and reverse reads from both ends of a double-stranded DNA. The final steps of most shotgun sequencing strategies involve sequencing from both ends of selected M13 clones to build a scaffold (map) and fill in map gaps using directed reads Chen et al., 1993). The two-color/four-lifetime strategy could be effectively used here for front- and back-end reads in a single lane, where eight probes need to be analyzed simultaneously. Sequencing from both ends also yields important positional information since the distance between the read pair is known (Edwards and Caskey, 1991).

As multiplexed dye systems using color discrimination are further developed, lifetime identification methods could be incorporated into any color method to further increase the multiplexing. The potential extension of the hybrid strategy may also involve the use of FRET systems. For example, a measurement of four different fluorescence lifetimes in four spectrally distinct channels that use FRET would allow 16 different parameters to be measured in a single lane and still, using a single excitation source, efficiently excite the donor dye. Such a dye set, however, remains to be developed.

0 0

Post a comment