Frequently Asked Questions
- RSE is able to selectively isolate genomic target segments that are typically an order of magnitude larger (20 kb) than fragments produced by other target enrichment methods. Long DNA sequence reads can greatly simplify the assembly of complex genomes, in particular if the target region contains difficult sequence elements.
- RSE requires only a small number of short (20-25 base) capture primers, typically spaced at distances of 3-8 kb, to pull down an extended region of interest. This makes it possible to enrich a region of interest even from potentially highly variable samples by creating a standard capture primer set based on known, conserved sequence elements.
- RSE allows the identification of regions with unknown sequence around each capture point. It can therefore generate accurate and complete sequence information even for complex regions of interest for which no reliable reference genomes exist. Other target enrichment methods are unable capture a broad genomic context.
600 ng – 6 µg per extraction are recommended depending on the project. DNA concentration should be at least 50 ng/µl.
- For small target regions (50-250 kb) and a low number of capture primers (10-50), we recommend an average distance between neighboring primers of 3-5 kb.
- For extended target regions (1-5 Mb) requiring a large number of capture primers (300+), the average distance between neighboring primers should be increased to 8-10 kb. This helps ensure adequate capture across the target region by retaining a sufficient capture primer concentration with a low risk for primer dimer formation.
For the typical design of a primer set, the region of interest is first repeat-masked to identify unique sequence elements across the target region that can serve as capture points. These sequences should then be reviewed for the possible presence of known mutations, which can either be avoided, incorporated or exploited during capture primer design.
- The 5’-end of a primer can be allowed to partly overlap repetitive sequence as long as its 3’-end is unique.
- The presence of a know polymorphism under the primer can be accepted by designing primers for both variants.
- If an allelic discrimination is desired, a known polymorphism can be positioned at the 3’-end of a capture primer (= HSE). In this case the enzymatic biotin labeling step and subsequent capture will only occur for primers whose 3’-ends match the targeted allele but not for variants that create a 3’-mismatch with the primers.