GAP-Seq: a method for identification of DNA palindromes

Abstract

Background: Closely spaced long inverted repeats, also known as DNA palindromes, can undergo intrastrand annealing to form DNA hairpins. The ability to form these hairpins results in genome instability, difficulties in maintaining clones in Escherichia coli and major problems for most DNA sequencing approaches. Because of their role in genomic instability and gene amplification in some human cancers, it is important to develop systematic approaches to detect and characterize DNA palindromes.

Results: We developed a new protocol to identify palindromes that couples the S1 nuclease treated Cot0 DNA (GAPF) with high-throughput sequencing (GAP-Seq). Unlike earlier protocols, it does not involve restriction enzymatic digestion prior to DNA snap-back thereby preserving longer DNA sequences. It also indicates the location of the novel junction, which can then be recovered. Using MCF-7 breast cancer cell line as the proof-of-principle analysis, we have identified 35 palindrome candidates and physically characterized the top 5 candidates and their junctions. Because this protocol eliminates many of the false positives that plague earlier techniques, we have improved palindrome identification.

Conclusions: The GAP-Seq approach underscores the importance of developing new tools for identifying and characterizing palindromes, and provides a new strategy to systematically assess palindromes in genomes. It will be useful for studying human cancers and other diseases associated with palindromes.

Figure 1

High-throughput next generation sequencing of fast annealing DNA treated by S1 nuclease. (A) Sequencing library construction. We prepared samples for sequencing based on efficient intrastrand base pairing of palindromes. Briefly, the genomic DNA was denatured and rapidly reannealed. After single-strand specific S1 nuclease digestion, DNA was extracted by phenol-chloroform and libraries were prepared for 454 and Illumina sequencing. (B) Data analysis. 1: Mapping of unique reads to human reference genome (NCBI36/hg18). Black lines represent mapped unique reads. Hatched rectangles represent regions masked by RepeatMasker (M). 2: Assembly of uniquely mapped reads to identify contiguous regions (contigs, black rectangles), and calculation of base read ratio (B) of each contig. For a contig region “c” with n mapped reads, . 3: Clustering two or more contigs with a base read ratio >1.5 that are within 7.5 kb of each other to make a joined contig. 4: Determining Rank “R” that is the sum of all read lengths in the joined contig divided by the length of the joined contig minus the masked regions .

Figure 2

Palindrome mapping strategy. (A) Read density distribution in Chr15q21.1: 47,529,204-47,550,373 region shown as 1 kb bins. (B) qPCR analysis to monitor for palindrome enrichment and determine the directionality of the Chr15q21.1 palindrome. We calculated the amount of depletion of a specific TaqMan primer set region based on Ct value before and after GAPF protocol in both IMR-90 and MCF-7 samples. The fold enrichment is based on comparing the fold depletion among different primer sets (P1, P2, P3 and P4) relative to a single copy sequence in the genome (RAD52). The location of TaqMan primer sets P1, P2, P3 and P4 is indicated in Figure 2C. (C) Map of genomic region Chr15: 47,520,000-47,550,000 with restriction sites and primer locations. (D) Southern blot analysis. Genomic DNA IMR-90 and MCF-7 cells was digested with BamHI, BglII or NcoI. Asterisks (*) mark the rearranged bands from MCF-7 genomic DNA. (E) Snap-back (SB) southern blots of BamHI digested IMR-90 and MCF-7 DNA. Arrowhead indicates the half sized fragments after snap-back in MCF-7. (F) Inversion-PCR. Three primers all from the same strand in normal genomic DNA were used for PCR (Primers 1–3). Since primers 1 and 2 are located in the palindromic region, they can also be used as reverse primers. Because primer 3 is in the spacer, it is able to produce a PCR product with primer 1 or 3 containing the novel junction “J” as indicated in the figure.

Figure 3

Schematics and sequence of palindrome junctions. Sequence analysis of the palindromic junctions identified 7 novel junctions. In each of the aligned breakpoint sequences, lowercase letters are Repeat-masker masked sequences. Uppercase letters represent unique sequences. Microhomology at the breakpoints is shown as bold letters. Insertion of Chr16 fragment at Chr15q21.1 spacer is shown as a black rectangle and deletion at Chr8q21.2 spacer is shown as a triangle in the schematics.

Figure 4

Summary of Affymetrix SNP6 copy number gains and palindrome candidates from Roche 454 sequencing of MCF-7. Key: red triangle-palindrome candidates from Roche 454 sequencing; green line-copy number gains based on Affymetrix SNP6 analysis; blue rectangle-centromere region. The data used to generate this figure are shown in Additional file 4: Table S3.