Xdrop™ is the perfect upstream enrichment method for targeted single molecule sequencing.
In “difficult to sequence” regions such as regions plagued with high GC-content, repeats and homopolymers, many DNA polymerases dissociate from the template DNA or create errors by slippage and other mechanisms.
The Samplix Xdrop™ platform, built on droplet-based enrichment and amplification technology, works equally well for all genomic regions including the “difficult to sequence” regions.
After Xdrop™ enrichment and amplification, long read library construction can be performed and samples sequenced with single molecule sequencing technologies. The targeted approach provides freedom to either increase read depth for individual samples or multiplexing of more samples on each sequencing chip.
Unknown regions or genome gaps are common challenges in de-novo sequencing but also occurs in relatively well characterized genomes when standard technologies for sample- and library preparation fail. Furthermore, gaps are poorly handled bioinformatically by short read sequencing mapping algorithms. Often, repeat regions which cannot uniformly align are discarded during data processing.
In the Samplix Xdrop™ enrichment process, primers will be designed for a short known sequence located within 10-20 kb of the unknown target region and used to identify droplets with long DNA molecules that encompass the target region. Because Xdrop™ captures very long DNA molecules, flanking regions of 20 kb on each side of the primer site will be enriched and amplified.
After isolation of the long DNA molecules, long read sequencing technologies can be used to provide sequence information from the neighboring unknown regions. Other examples with unknown sequence context are genomic integration sites for viruses, transposons and other mobile genetic elements. Xdrop™ can also be used for QC of transgenes in genomes edited with CRISPR and viral vectores. In these instances the unknown sequence context can for example be integration site confirmation or validation but also off target investigation.
Haplotype phasing can be a very difficult process, especially across longer genomic distances. With use of Xdrop technology from Samplix, combined with downstream single molecule sequencing, you are now able to maintain important locus information even if a locus contains “difficult to sequence regions”. The long DNA molecules will maintain the phasing information throughout enrichment and amplification steps. Combined with downstream single molecule sequencing, Xdrop™ will provide quicker and more accurate phasing information than any other available technology.
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