ABSTRACT
BACKGROUND: Retroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (< 1%) subclonal cancer specific RE insertions is complicated. RESULTS: Here we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions. CONCLUSIONS: This study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.
ABSTRACT
BACKGROUND: There is increasing evidence that the transpositional activity of retroelements (REs) is not limited to germ line cells, but often occurs in tumor and normal somatic cells. Somatic transpositions were found in several human tissues and are especially typical for the brain. Several computational and experimental approaches for detection of somatic retroelement insertions was developed in the past few years. These approaches were successfully applied to detect somatic insertions in clonally expanded tumor cells. At the same time, identification of somatic insertions presented in small proportion of cells, such as neurons, remains a considerable challenge. RESULTS: In this study, we developed a normalization procedure for library enrichment by DNA sequences corresponding to rare somatic RE insertions. Two rounds of normalization increased the number of fragments adjacent to somatic REs in the sequenced sample by more than 26-fold, and the number of identified somatic REs was increased by 8-fold. CONCLUSIONS: The developed technique can be used in combination with vast majority of modern RE identification approaches and can dramatically increase their capacity to detect rare somatic RE insertions in different types of cells.
