We inserted a tag into the genome of human cells and made clonal cell lines. We can tell by PCR that most of the clones have an insert that is much larger than expected (2-3kb instead of 700bp), and we want to find out what exactly was inserted there (is it part of our plasmid? is it just repeated tags?...?). We were unable to Sanger the whole large amplicon because it appears that at least some of the sequence is repetitive and therefore we can't identify unique primers to walk along it.
Will this be NGS-friendly? I'm envisioning maybe sonicating the large amplicon and then using random hexamers to ligate adapters. Is that necessary? is there an easier way? If it really is a repetition of our tag, I assume aligning the reads is going to be a challenge all in it's own.
Will this be NGS-friendly? I'm envisioning maybe sonicating the large amplicon and then using random hexamers to ligate adapters. Is that necessary? is there an easier way? If it really is a repetition of our tag, I assume aligning the reads is going to be a challenge all in it's own.
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