Abstract
Microbial cloning makes Sanger sequencing of complex DNA samples possible but is labor intensive. We present a simple, rapid and robust method that enables laboratories without special equipment to perform single-molecule amplicon sequencing, although in a low-throughput manner, from sub-picogram quantities of DNA. The method can also be used for quick quality control of next-generation sequencing libraries, as was demonstrated for a metagenomic sample.
Highlights
Sanger sequencing has long been, and still is, the most used method for DNA sequencing
Applying Sanger sequencing on complex sample materials commonly requires an initial microbial sub-cloning step, which is often used for validating next-generation sequencing libraries [2]
Generation of polonies According to the Poisson distribution, sample concentration should be kept low to achieve a high proportion of single-molecule polonies among all (Table S1 and Figure S1)
Summary
Sanger sequencing has long been, and still is, the most used method for DNA sequencing. Applying Sanger sequencing on complex sample materials commonly requires an initial microbial sub-cloning step, which is often used for validating next-generation sequencing libraries [2].
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