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Sequencing multimegabase-template DNA with BigDye terminator chemistry
C R Heiner1, K L Hunkapiller, S M Chen
1Advanced Center for Genetic Technology, PE-Applied Biosystems, Foster City, California 94404, USA.
Genome Research
|May 20, 1998
Summary
Directly sequencing large microbial genomes using BigDye terminators and custom primers on automated instruments yields long reads. This method efficiently fills gaps in genome projects and sequences targeted regions, bypassing subcloning.
Area of Science:
- Genomics
- Molecular Biology
- Bioinformatics
Background:
- Automated DNA sequencing is crucial for genomics.
- Sequencing large microbial genomes presents challenges.
- Direct sequencing methods can improve efficiency.
Purpose of the Study:
- To present cycle sequencing conditions for large-template microbial DNA using BigDye terminators.
- To demonstrate the utility of this method for gap closure and targeted sequencing.
Main Methods:
- Utilized BigDye terminators for cycle sequencing.
- Employed custom primers for direct sequencing of microbial genomic DNA.
- Applied automated sequencing instruments.
Main Results:
- Successfully sequenced large microbial genomes (e.g., Escherichia coli, Streptococcus pneumoniae).
- Achieved average read lengths exceeding 700 bp from unique primer sites.
- Demonstrated sufficiency of read lengths for closing final gaps in genome projects.
Conclusions:
- Direct sequencing with BigDye terminators is effective for large microbial genomes.
- The method simplifies gap filling and targeted sequencing, reducing manipulation steps.