Illumina mate-paired DNA sequencing-library preparation using Cre-Lox recombination

Filip Van Nieuwerburgh, Ryan C. Thompson, Jessica Ledesma, Dieter Deforce, Terry Gaasterland, Phillip Ordoukhanian, Steven R. Head

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

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 3kb 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.

Original languageEnglish
Pages (from-to)e24
JournalNucleic Acids Research
Volume40
Issue number3
DOIs
StatePublished - Feb 2012
Externally publishedYes

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