Fig. 1.

Schematic workflow for completion of draft bacterial genomes using long-read sequencing of synthetic genomic pools. Individual bacterial genomes are sequenced using a miniaturized, cost effective, multiplexed sequencing protocol on the Illumina platform. Short Illumina reads are used for de novo assembly of draft bacterial genomes (Illumina contigs). The gDNA of bacterial isolates are then combined into a synthetic genomic pool library (~80Mbp total genome size) and subjected to standard PacBio sequencing without multiplexing. Generated long reads are mapped to Illumina assemblies for sorting high-quality long reads of individual genomes. This is followed by de novo long-read assembly to generate ultra-long PacBio contigs for each genome, and finally, completion of draft bacterial genomes by hybrid assembly of Illumina short reads, PacBio long reads, and ultra-long PacBio contigs. The final assembly is polished by high-quality Illumina reads to correct potential assembly errors. Names of bioinformatics software used at each step of the assembly pipeline are indicated in parenthesis