Optimization of protocol for sequencing of difficult templates

Abstract

In this paper, we have fine-tuned a DNA sequencing protocol suitable for a wide range of difficult templates. The primary goal was to evaluate a number of parameters--such as various dye terminator mixes in the presence or absence of additives, the amount of DNA or primer, and cycling protocols--about the effectiveness of reading through complex regions. We showed that the modification of a published protocol leads to significant (75%) cost reduction without forfeiting quality of the data. In the recommended protocol, we used betaine as a standard additive, but better results can be obtained when betaine and Reagent A are mixed in an equivalent ratio.

Keywords: additives; complex DNA regions; dGTP.

FIGURE 1

Examples of Q ≥ 20 read length (RL) values for different dye dilution strengths in the presence or absence of betaine (Bet). (A) DNA #1 sequenced with forward primer. (B) DNA #5 sequenced with reverse primer. (C) DNA #6 sequenced with forward primer. FS, Full-strength of dye or dye mixes; 2, 4, 8× dil = dye or dye mixes diluted two-, four-, or eightfold, respectively.

FIGURE 2

Effect of amount of DNA (A) or primer (B) on Q ≥ 20 read-length values. Four different templates were sequenced at three levels of DNA or primer. Template #3 was sequenced in both directions, as this is the most difficult template in this series.

FIGURE 3

Testing various cycling conditions on six different DNAs with forward (A) or reverse (B) primers.

FIGURE 4

Comparison of data quality using cycling Protocol #1 (A) or Protocol #5 (B). Note that the amount of dark-blue bases is higher (and starts earlier) in the example sequenced with Protocol #5. In Sequencher's notation, the light-blue color indicates bases with Q ≥ 20. Darker blue colors of bases indicate their poorer quality.