Multiplexed Non-barcoded Long-Read Sequencing and Assembling Genomes of Bacillus Strains in Error-Free Simulations.

The generation of genomic data from microorganisms has revolutionized our abilities to understand their biology, but it is still challenging to obtain complete genome sequences of microbes in an automated high-throughput and cost-effective manner. While the advent of second-generation sequencing technologies provided significantly higher throughput, their shorter lengths and more pronounced sequence-context bias led to a shift towards resequencing applications. Recently, single molecule real-time (SMRT) DNA sequencing has been used to generate sequencing reads that are much longer than other sequencing platforms, facilitating de novo genome assembly and genome finishing. Here we introduced a novel multiplex strategy to make full use of the capacity and characteristics of SMRT sequencing in microbe genome assembly. We used error-free simulations to evaluate the practicability of assembling SMRT genomic sequencing data from multiple microbes into finished genomes once at a time. Then we compared the influence of two key factors, including sequencing coverage and read length, on multiplex assembling. Our results showed that long-read genomic sequencing inherently provided the ability to assemble genomic sequencing data from multiple microbes into finished genomes due to its long length. This approach might be helpful for the various groups of microbial genome projects or metagenomics research.