RESUMO
We synthesized reversible terminators with tethered inhibitors for next-generation sequencing. These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides, and we used them to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99% of the region sequenced. This single-molecule approach generated high-quality sequence data without the need for target amplification and thus avoided concomitant biases.
Assuntos
Cromossomos Artificiais Bacterianos/química , DNA/química , Nucleotídeos/química , Análise de Sequência de DNA/métodos , Animais , Cromatografia Líquida de Alta Pressão , Cromossomos Artificiais Bacterianos/genética , Simulação por Computador , Cães , Nucleotídeos/genética , Sensibilidade e Especificidade , Especificidade por SubstratoRESUMO
The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.