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Vol. 8, Issue 5, 557-561, May 1998
GENOME METHODS
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ABSTRACT |
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Top
Abstract
Introduction
Results & Discussion
Methods
References
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Using the recently introduced BigDyeTM terminators, large-template DNA can be directly sequenced with custom primers on automated instruments. Cycle sequencing conditions are presented to sequence DNA samples isolated from a number of microbial genomes including 750-kb Ureaplasma urealyticum, 1.2-Mb Mycoplasma fermentans, 2.3-Mb Streptococcus pneumoniae, and 4.6-Mb Escherichia coli. Average read lengths of >700 bp from unique primer annealing sites are often sufficient to fill final gaps in microbial genome sequencing projects without additional manipulations of template DNA. The technique can also be applied to sequence-targeted regions, thereby bypassing tedious subcloning steps.
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INTRODUCTION |
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Top
Abstract
Introduction
Results & Discussion
Methods
References
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In microbial genome or large-insert clone sequencing projects that
use the predominant random subclone sequencing strategy, progress tends
to decrease dramatically at late stages as one confronts
gaps. At these points, DNA is under-represented or
unstable in subclones (E.Y. Chen et al. 1996
; Chissoe et al. 1997).
Further sequencing with additional random subclones is then inefficient at best, and one must frequently employ alternative cloning systems or
additional methods like long-range PCR to recover missing DNA (C.N.
Chen et al. 1996). The variability of performance of these methods and
the necessity for custom-tailored work tend to hamper the late stages
of sequencing efforts. In contrast, if one can sequence directly from
genomic DNA (or large-insert clones such as BACs or PACs) with walking
primers, cumbersome work to fill gaps could be completed in a much
shorter time.
As an example, in a recent project to sequence the 750-kb genome of
Ureaplasma urealyticum (J. Glass, in prep.) assemblage of
~13,000 sequence reads and combinatorial PCR reactions to join contigs left two gaps. No 
pUC, or M13 subclones were recovered that spanned the gaps, nor were PCR products derived with any of
several sets of flanking primers. The difficulty of cloning these
segments is probably attributable to repeated sequences in and near the
two gaps, but the high sensitivity of the recently introduced BigDye
terminator (Rosenblum et al. 1997) permitted direct sequencing of the
gap regions on genomic U. urealyticum DNA templates. Using the
conditions described in this report, two gaps of 259 and 121 bp were
sequenced from both strands with walking primers to complete the
project of 751,723 bp.
Direct sequencing was further tested for larger templates, and good results were reproducibly obtained with 1.2-Mb Mycoplasma fermentans, 2.3-Mb Streptococcus pneumoniae, and 4.6-Mb Escherichia coli genomic DNA (see example in Fig. 1). In addition, several difficult gaps in sequencing projects with BAC clones, ranging in size from 140 to 250 kb, have also been filled in this manner. Essentially the method is applicable whenever 2-3 µg of high-quality large-template DNA is available.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Results & Discussion
Methods
References
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Figure 1 shows an example of the results from these experiments.
Although the signal intensity tends to be low
only ~10%-20% compared to the data from regular M13 or pUC templatesbase-calling quality remains high, because the baseline noise is sharply reduced by
the increased brightness and improved spectral resolution of the BigDye
terminators (Rosenblum et al. 1997). Lower signal strength is expected
considering the molarity of microbial template DNA, which is several
hundred to a thousand times less than that of the regular plasmid
templates. Higher level of primers (2×-5×) and greater number of
cycles (from 45 to 60, more cycles for larger templates) as described
in Methods helped to boost the signal intensities. The addition of
cycles (up to 99) has been found to increase the signal strength and
decrease the readable range (see Table 1). Accurate
quantitation of template DNA to within 2-4 µg is essential. Too
much template (>5 µg) produced much lower quality results (see
Fig. 2 for an example), whereas too little DNA also gave rise to weak
signal and low-quality results (data not shown).
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There are several other factors that are also important for optimal results. First, template DNA should be free of salts, detergents, proteins, and cell debris that might interfere with the primer annealing. Second, as may be expected, having a good primer is critical. Successful primers have been typical 21-25-mers with appropriate GC contents from a unique site. Only 1 of the 10 primers tested failed to generate a useful sequence because that primer annealed at two different locations on the S. pneumoniae genome. Third, special care should be taken in the elimination of excess dye terminators before loading samples on gels (note that carryover of dyes can be seen in Fig. 1 in the region of bases 45-55; apparently the system is very sensitive to residual dye when signals are so low). Fourth, to get high-quality, low-signal data, it is important to have a well-tuned sequencing instrument equipped with a good multicomponent matrix and base-calling software capable of analyzing data with weak signals. Here we used version 3.0.1b3 software, which requires no minimal signals.
There appears to be no correlation between the quality of the sequence
data and the size of the template, as shown in Table 2, in which nine
different sequences obtained from bacterial genomic templates are
compared with corresponding GenBank sequences that were presumably
derived from subcloned templates. Using the unedited
sequence files of up to 600 bases beyond the first legible base,
genome-derived sequences have an average fidelity of 98.2% (1.1%
N and 0.7% discordant base-calls). After minimal manual editing of the sequences, the average useable read length was 712 ± 18 bases. It is interesting that the signal strength remains relatively constant among these nine genomic templates ranging from
0.75 to 4.6 Mb. This suggests that the present protocol, after some
modifications, may be applicable to even larger templates. The method,
nevertheless, does require 2-3 µg of high-quality template DNA for
each sequencing reactionan amount that could be difficult to obtain
from certain microorganisms.
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In summary, we have demonstrated that template DNAs up to 4.6 Mb in size can be directly sequenced with automated sequencers using custom primers and BigDye terminators. This protocol could expedite large-scale sequencing projects by facilitating gap closure, especially for difficult areas that are refractory to cloning and PCR methods. The technique can also be applied to bypass tedious subcloning steps in bacterial sequencing projects that focus on individual genes.
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METHODS |
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Top
Abstract
Introduction
Results & Discussion
Methods
References
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Preparation of DNA Samples
Multiple methods, including Easy-DNA kit (Invitrogen, USA),
SDS-proteinase K lysis procedure, and isopycnic banding in CsCl gradients (Wilson 1994), all worked well to prepare microbial genomic
DNA samples for sequencing. BAC DNA was purified as described by
Rosenblum et al (1997). Sequencing primers were designed using Oligo
5.0 (National Biosciences, USA). Sizes varied from 21 to 25 bases and
Tm from 60°C to 74°C (GC% method).
Sequencing Conditions
Each cycle sequencing reaction contained 16 µl of BigDye Terminator mix (PE-Applied Biosystems, USA), 13 pmoles of primer, and 2-3 µg of microbial DNA (or 6 pmoles of primer and 0.4 µg of BAC DNA) in a total of 40-µl volume. In some experiments 1 µl of ThermoFidelase I (Fidelity Systems, USA) was included to improve data quality, but the results were inconsistent, so it is not a standard component of current reaction mixtures. The cycle conditions were initial denaturation at 95°C for 5 min followed by 30-60 cycles (30 for BACs, 45 for all microbial DNAs, except E. coli samples, which get 60 cycles; see Results/Discussion) at 95°C for 30 sec, 55°C for 20 sec, and 60°C for 4 min. Excess dye terminators were removed with a spin column (PE-Applied Biosystems User Manual) and reaction mixtures were dried in a SpeedVac system. Each sample was resuspended in 2 µl of formamide solution and denatured by heat, and the entire volume loaded on an ABI 377 automated DNA sequencing instrument with a 36-well comb and a 48-cm WTR (well-to-read) gel. Electrophoresis was at 2400 V for 10-11 hours. Sequence data were analyzed by ABI version 3.0.1b3 software modified for weak signal base-calling (available through PE-ABI ftp site).
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ACKNOWLEDGMENTS |
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We thank Dan Allison for modifying the base-calling software, Jennifer Glass for help in preparing the microbial DNAs, and Chun-Nan Chen for valuable discussions. This work is supported, in part, by the National Institutes of Health (grants HG00201 and RO1 AI28279).
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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FOOTNOTES |
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4 Corresponding author.
E-MAIL cheney{at}perkin-elmer.com; FAX (650) 638-6177.
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REFERENCES |
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Top
Abstract
Introduction
Results & Discussion
Methods
References
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Received November 26, 1997; accepted in revised form March 18, 1998.
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