Facilitated sequence assembly using densely labeled optical DNA barcodes: A combinatorial auction approach

Abstract

The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard “foreign” contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.

Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak

“…Complementing our analysis with long-read PacBio sequencing of key isolates demonstrated that the overlap between experimental and theoretical barcodes (15, 19) was excellent. This comparison confirmed that the location of the bla CTX-M-15 gene along the barcode of the smaller plasmid, predicted by Cas9 restriction, was correct.…”

The resistomes of six carbapenem-resistant pathogens – a critical genotype–phenotype analysis

“…Theoretical optical DNA maps from assembled DNA sequences were created similar to Nilsson et al . [17] with updated ligand binding constants [19]. Comparisons between theoretical and experimental optical DNA maps were performed as described previously [14, 17].…”

Enzyme-free optical DNA mapping of the human genome using competitive binding

“…Bacterial Artificial Chromosomes (BACs) were used to optimize the experimental settings and the P -value threshold (37,39). For details about the five BACs used in the study, see Supplementary Table S1.…”

“…To compare an experimental barcode to the human genome, theoretical barcodes based on the base pair sequence of all 24 chromosomes (GRCh38.p7 dataset, retrieved from NCBI) were generated, using previously established methods (36,37). In short, the theoretical barcode is estimated based on sequence-dependent binding constants of netropsin, a sequence-independent binding constant of YOYO-1, and the point-spread function of the microscope.…”

“…Netropsin blocks AT-rich regions from binding of the fluorescent, nonspecific, YOYO-1, which leads to that AT-rich regions are darker than GC-rich regions (Figure 1) (35). We have previously used this competitive binding-based assay for studies on bacterial DNA (36), and in particular bacterial plasmids (37–42). The principle is the same as the melt mapping assay initially proposed by Reisner et al.…”