Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing

Abstract

Advances in sequencing technology have drastically increased the depth and feasibility of bacterial genome sequencing. However, little information is available that details the specific techniques and procedures employed during genome sequencing despite the large numbers of published genomes. Shotgun approaches employed by second-generation sequencing platforms has necessitated the development of robust bioinformatics tools for in silico assembly, and complete assembly is limited by the presence of repetitive DNA sequences and multi-copy operons. Typically, re-sequencing with multiple platforms and laborious, targeted Sanger sequencing are employed to finish a draft bacterial genome. Here we describe a novel strategy based on the identification and targeted sequencing of repetitive rDNA operons to expedite bacterial genome assembly and finishing. Our strategy was validated by finishing the genome of Paenibacillus polymyxa strain CR1, a bacterium with potential in sustainable agriculture and bio-based processes. An analysis of the 38 contigs contained in the P. polymyxa strain CR1 draft genome revealed 12 repetitive rDNA operons with varied intragenic and flanking regions of variable length, unanimously located at contig boundaries and within contig gaps. These highly similar but not identical rDNA operons were experimentally verified and sequenced simultaneously with multiple, specially designed primer sets. This approach also identified and corrected significant sequence rearrangement generated during the initial in silico assembly of sequencing reads. Our approach reduces the required effort associated with blind primer walking for contig assembly, increasing both the speed and feasibility of genome finishing. Our study further reinforces the notion that repetitive DNA elements are major limiting factors for genome finishing. Moreover, we provided a step-by-step workflow for genome finishing, which may guide future bacterial genome finishing projects.

Isolation, identification and characterization of Paenibacillus polymyxa CR1 with potentials for biopesticide, biofertilization, biomass degradation and biofuel production

“…Phylogenetic analysis was based on the 16S rRNA gene sequenced in this work and deposited in Genbank (accession no. KF620436.1, the isolate’s full genome has since been sequenced [ 20 – 22 ]). The 16S rRNA gene was aligned against the NCBI nucleotide database using Blastn, showing 99 % identity to sequences from P. polymyxa strains E681 (CP000154.1), SC2, and M1.…”

Whole Genome Mapping with Feature Sets from High-Throughput Sequencing Data

“…Optical mapping and nanochannel genome mapping can be used to generate whole genome ordered restriction/specific sequence motif maps directly using genomic DNA [ 41 , 42 ] and, in this case, have multiple advantages, such as omitting cloning of large insert DNA fragments, providing potentially very long scaffolds and discerning genome wide DNA methylation profiles [ 43 – 45 ]. However, these maps are not determinative and cannot provide DNA templates for sequence completion.…”

Actinobacteria diversity associated with sponge Amorphinopsis exacavans from Rancabuaya Beach, Garut, Indonesia

“…Because of this, Q30 is regarded as a quality standard in NGS. A good sequencing run for most Hiseq platforms for metagenomics should produce at least 70-80% of the Q30 data [20]. The main family, genus, and species were Staphylococcaceae, Staphylococcus, and Staphylococcus aureus, according to inner tissue sponges (endosymbionts).…”