Genomic representations using concatenates of Type IIB restriction endonuclease digestion fragments

Abstract

We have developed a method for genomic representation using Type IIB restriction endonucleases. Representation by concatenation of restriction digests, or RECORD, is an approach to sample the fragments generated by cleavage with these enzymes. Here, we show that the RECORD libraries may be used for digital karyotyping and for pathogen identification by computational subtraction.

Figure 1

BsaXI digested human genomic DNA with 10 bp ladder (Invitrogen). Acrylamide gel (8%), stained with GelStar (Cambrex).

Figure 2

Overview of method for the construction of Type IIB restriction enzyme tag libraries.

Figure 3

The results of a simulation study analyzing number of sequenced tags necessary to detect various types of alterations, as a function of alteration length. For each of the four types of alterations shown, digital karyotyping experiments were simulated for a range of alteration lengths. These simulations were conducted by randomly sampling from a set of integers representing the in silico tags, with the randomly placed altered region simulated by increasing (for amplification) or decreasing (for deletion) the integer value representing the number of copies of the corresponding tags. Simulations were repeated 100 times for each combination of alteration type and length, and isomeric curves were fit to 90% detection rates for each alteration type.

Figure 4

Predicted copy numbers using SNP arrays (top panel) and RECORD libraries (bottom panel). Arrows indicated PCR-confirmed deletions of the ROBO1 gene (base position ∼79.1 Mb in chromosome 3) and the CDKN2A tumor suppressor gene (base position ∼22.0 Mb in chromosome 9) and a PCR-confirmed amplification of the region containing the MYC oncogene (base position ∼128.7 Mb in chromosome 8).

Figure 5

Vectorette PCR. A 2% agarose gel stained with GelStar. First lane upper and lower panel: 100 bp ladder (Invitrogen). Upper panel: lanes 2–13, six sets of EBV tag-specific primers (HCC38 and HCC38 BL). Lower panel: lanes 2–13, six sets of primers designed from non-matching tags (HCC38 and HCC38 BL).