Contig-Layout-Authenticator (CLA): A Combinatorial Approach to Ordering and Scaffolding of Bacterial Contigs for Comparative Genomics and Molecular Epidemiology

<div><p>A wide variety of genome sequencing platforms have emerged in the recent past. High-throughput platforms like Illumina and 454 are essentially adaptations of the shotgun approach generating millions of fragmented single or paired sequencing reads. To reconstruct whole genomes, the reads have to be assembled into contigs, which often require further downstream processing. The contigs can be directly ordered according to a reference, scaffolded based on paired read information, or assembled using a combination of the two approaches. While the reference-based approach appears to mask strain-specific information, scaffolding based on paired-end information suffers when repetitive elements longer than the size of the sequencing reads are present in the genome. Sequencing technologies that produce long reads can solve the problems associated with repetitive elements but are not necessarily easily available to researchers. The most common high-throughput technology currently used is the Illumina short read platform. To improve upon the shortcomings associated with the construction of draft genomes with Illumina paired-end sequencing, we developed Contig-Layout-Authenticator (CLA). The CLA pipeline can scaffold reference-sorted contigs based on paired reads, resulting in better assembled genomes. Moreover, CLA also hints at probable misassemblies and contaminations, for the users to cross-check before constructing the consensus draft. The CLA pipeline was designed and trained extensively on various bacterial genome datasets for the ordering and scaffolding of large repetitive contigs. The tool has been validated and compared favorably with other widely-used scaffolding and ordering tools using both simulated and real sequence datasets. CLA is a user friendly tool that requires a single command line input to generate ordered scaffolds.</p></div

Schematic overview of CLA pipeline.

<p>The schema of CLA pipeline is divided into three stages. In the first stage, a reference based order is derived followed by the second stage where connections between the individual contigs are extracted based on alignment information. The final stage makes use of the information from the first two stages to decide the final order followed by scaffolding and gap filling.</p

Comparative statistics of CLA and Scaffolding tools generated using real dataset.

<p>Comparative statistics of CLA and Scaffolding tools generated using real dataset.</p

Defining the start and end of a contig based on the insert size.

<p>Connectivity between two consecutive contigs is decided by the paired reads from a single insert. To find such connections between contigs, each contig is theoretically divided into start, mid and end regions based on the insert size.</p