Sequencing the unknown region of a large amplicon

[amanda_h]

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.

[nucacidhunter]

You can use PacBio RSII or Sequel to directly sequence whole amplicon with high accuracy.

[GenoMax]

How many clones are you planning to sequence?

PacBio option (while good) may be expensive. You can see if anyone has a nanopore sequencer available nearby. While the error rate is a problem it should give you some qualitative idea of what you are looking for.

[pmiguel]

I hate Sanger sequencing and wish it were no longer needed and could be kicked to the curb! That said, with current technology, this is a job for Sanger sequencing. Clone your PCR fragment. Sequence in from the ends and look for a propitious restriction enzyme site you can delete part of the proximal insert on one side with. Do the same for the other, if necessary.

This brings the unique sequencing primer site closer to the middle of your PCR fragment and allows you to obtain the sequence from it.

Alternatively you could, after cloning, use a transposon insertion kit (EG, epicentre EZ-Tn5) and sequence from the ends of the transposon. But that is probably overkill for a 3kb insert.

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Phillip