title: Illumina mate-paired DNA sequencing-library preparation using Cre-Lox recombination.
abstract: Standard Illumina mate-paired libraries are constructed from 3- to 5-kb DNA fragments by a blunt-end circularization. Sequencing reads that pass through the junction of the two joined ends of a 3-5-kb DNA fragment are not easy to identify and pose problems during mapping and de novo assembly. Longer read lengths increase the possibility that a read will cross the junction. To solve this problem, we developed a mate-paired protocol for use with Illumina sequencing technology that uses Cre-Lox recombination instead of blunt end circularization. In this method, a LoxP sequence is incorporated at the junction site. This sequence allows screening reads for junctions without using a reference genome. Junction reads can be trimmed or split at the junction. Moreover, the location of the LoxP sequence in the reads distinguishes mate-paired reads from spurious paired-end reads. We tested this new method by preparing and sequencing a mate-paired library with an insert size of 3 kb from Saccharomyces cerevisiae. We present an analysis of the library quality statistics and a new bio-informatics tool called DeLoxer that can be used to analyze an IlluminaCre-Lox mate-paired data set. We also demonstrate how the resulting data significantly improves a de novo assembly of the S. cerevisiae genome.
authors: Laboratory of Pharmaceutical Biotechnology, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.
authors: Filip
authors: F
authors: Van Nieuwerburgh
authors: Ryan C
authors: RC
authors: Thompson
authors: Jessica
authors: J
authors: Ledesma
authors: Dieter
authors: D
authors: Deforce
authors: Terry
authors: T
authors: Gaasterland
authors: Phillip
authors: P
authors: Ordoukhanian
authors: Steven R
authors: SR
authors: Head
mesh_terms: D016681:Genome, Fungal; D015698:Genomic Library; D019426:Integrases; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D012984:Software
chemical_list: C045073:Cre recombinase; D019426:Integrases