LTC: a novel algorithm to improve the efficiency of contig assembly for physical mapping in complex genomes

Abstract

BackgroundPhysical maps are the substrate of genome sequencing and map-based cloning and their construction relies on the accurate assembly of BAC clones into large contigs that are then anchored to genetic maps with molecular markers. High Information Content Fingerprinting has become the method of choice for large and repetitive genomes such as those of maize, barley, and wheat. However, the high level of repeated DNA present in these genomes requires the application of very stringent criteria to ensure a reliable assembly with the FingerPrinted Contig (FPC) software, which often results in short contig lengths (of 3-5 clones before merging) as well as an unreliable assembly in some difficult regions. Difficulties can originate from a non-linear topological structure of clone overlaps, low power of clone ordering algorithms, and the absence of tools to identify sources of gaps in Minimal Tiling Paths (MTPs).ResultsTo address these problems, we propose a novel approach that: (i) reduces the rate of false connections and Q-clones by using a new cutoff calculation method; (ii) obtains reliable clusters robust to the exclusion of single clone or clone overlap; (iii) explores the topological contig structure by considering contigs as networks of clones connected by significant overlaps; (iv) performs iterative clone clustering combined with ordering and order verification using re-sampling methods; and (v) uses global optimization methods for clone ordering and Band Map construction. The elements of this new analytical framework called Linear Topological Contig (LTC) were applied on datasets used previously for the construction of the physical map of wheat chromosome 3B with FPC. The performance of LTC vs. FPC was compared also on the simulated BAC libraries based on the known genome sequences for chromosome 1 of rice and chromosome 1 of maize.ConclusionsThe results show that compared to other methods, LTC enables the construction of highly reliable and longer contigs (5-12 clones before merging), the detection of "weak" connections in contigs and their "repair", and the elongation of contigs obtained by other assembly methods.

A Distorted Circadian Clock Causes Early Flowering and Temperature-Dependent Variation in Spike Development in the Eps-3Am Mutant of Einkorn Wheat

“…Additionally, BLAST searches were performed utilizing the existing sequence information; BAC-End Sequences and sequenced BACs (http://webblast.ipkgatersleben.de/barley/). Fingerprinted BACs were assembled by using the FPC software (http://www.agcol.arizona.edu/software/fpc/) and the obtained contig was verified by the LTC software (Frenkel et al 2010). The latter one allowed for more precise Minimum Tiling Path (MTP) selection as well as the exclusion of BACs wrongly assigned to the fingerprint-contig.…”

A Sequence-Ready Physical Map of Barley Anchored Genetically by Two Million Single-Nucleotide Polymorphisms

“…The linear topology contigs (LTC) tool (Frenkel et al, 2010) had been specifically designed for the construction and verification of physical maps of complex genomes. In particular, LTC is able to detect misassembled contigs, where the net of clone overlaps has branched or cyclical topological structures contradicting the linearity of the chromosomes are observed.…”

“…BAC contigs generated by FPC were further analyzed with the alternative physical mapping tool LTC (Frenkel et al, 2010) to trace misassembled contigs caused by chimerical clones and false clone overlaps. Misassemblies were detected by analysis of the topological structure of the net of clone overlaps within contigs (using a Sulston score cutoff of 1e-20): a contig was considered as putatively problematic if this net had a nonlinear structure.…”

“…To check the linearity of the net, LTC automatically selected one of the possible diametric paths (the longest nonreducible path going through edges corresponding to significantly overlapping clones, with vertices corresponding to clones) and scored the ranks of all vertices relative to the vertices from this diametric path (vertices from the diametric path were ranked as 0, vertices connected to vertices from the diametric path by a single edge were ranked as 1, etc.). The presence of vertices having a rank of 2 and more points indicated the nonlinearity of the net (Frenkel et al, 2010). In the case of nonlinearity of the net structure, LTC automatically tries to repeat the analysis using a more stringent cutoff (with increasing cutoff stringency by 1 order of magnitude at each step).…”

“…In many cases, this increasing stringency resulted in the exclusion of false overlaps, and the net of remaining clone overlaps was found to have a linear structure. To identify the presence of putative chimerical clones (composed by artificial fusion of DNA from distant genomic regions), the parallel clone overlaps were automatically searched for each clone of the contig (Frenkel et al, 2010). LTC was also used for the recalculation of clone end coordinates within FPC-based contigs (algorithms implemented in LTC enable reducing errors in the estimation of clone position and contig length caused by local optimization in FPC).…”