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.

Figure 1.

LoxP adapter oligos. Both double-stranded oligos have a 3-bp overhang to allow for directional ligation.

Figure 2.

Schematic of the Cre recombination-related library preparation steps. NNNNN denotes 2- to 5-kb DNA fragments taken into the LoxP adaptor ligation. LoxP sequences are in red and the 8-bp spacer between the two palindromic elements are in green. Orientation of the spacer region determines direction of recombination. Marked in yellow are the biotinylatedthymidines.

Figure 3.

Schematic of the classification scheme used by DeLoxer. The dark blue line represents the original circularized DNA fragment obtained by Cre recombination and polymerase fill-in, while the block represents a single linear piece of that fragment obtained during the mate-pair sequencing prep. The ends are sequenced (light blue), yielding a read pair that must be classified, while the center of the fragment may be unsequenced (light gray). The possible positions of the LoxP adapter relative to the sequenced fragment are shown as green boxes. Case I: the LoxP site aligns to the start of one read, and the overlap is trimmed off. This LoxP site is outside the two reads, so the read pair is not a mate pair and is labeled as ‘paired-end’. Case II: The LoxP site aligns near the center of one read. The read is discarded as it does not contain at least 36 contiguous bp of genomic DNA, and the other read in the pair is retained as an unpaired read. Case III: The LoxP site aligns to the end of one (or both) reads, and the overlap is trimmed off. This site lies between the two reads, making this pair a mate-pair. Case IV: The LoxP site lies entirely in the unsequenced center portion of the DNA fragment, and does not overlap either read, so the pair is LoxP-negative. Case V: The LoxP site does not occur anywhere within the sequenced fragment, so the pair is LoxP-negative. Note that although Case IV represents a mate-pair, it is indistinguishable from Case V.

Figure 4.

Fragment size distribution of (a) mate-paired reads, (b) paired-end reads and (c) LoxP-negative reads.