Sequencing repeats: how to?

[gaster]

Anyone know of a way to sequence repeats? We have a large 1000bp region of repeats. We can't sequence though it with either 454 or even ABI. Seems like there might some techniques out there for this?

Anyone?

Thanks,

Gaster

[nickloman]

Anyone know of a way to sequence repeats? We have a large 1000bp region of repeats. We can't sequence though it with either 454 or even ABI. Seems like there might some techniques out there for this?

Anyone?

Thanks,

Gaster

Tricky - you need to define a bit more clearly the nature and structure of the repeats (are they likely to be perfect, imperfect, tandem or separated, is the repeat 1000bp long or is it a kilobase of shorter repeats?). Paired-end reads are often helpful for repeat regions I believe 454 can use 3kb and 20kb inserts and Illumina offer short (250-500bp) and mid-range (2-5kb) inserts. But despite all this, some kinds of repeats are still 'impossible' to sequence.

[gaster]

Thanks for your reply. We think these are perfect repeats of 90 basepairs. It is just one region so we would rather not do another 454 or a Solexa run. Any ideas?

[nickloman]

Sounds like you have 11 or so tandem perfect repeats each 90 base pairs.

How many times do these regions occur in your genome?

If only once, you could try and use read depth (as mapped against a single copy of the repeat, or a repetitive contig as produced by Newbler) as a guide to the total number of copies.

Otherwise I think you will have difficulty using your existing data to get an answer as your repeats are longer than read length.

[westerman]

You might be able to get a 3730 to sequence over 1000 bases. We have had a couple hundred 1000+ base high quality base sequencing runs (out of thousands) over the last 10 years. Bribe your friendly sequencing center to take extra care in the sequencing run. It does depend on if your sequence has homopolymer segments within the repeats.

I agree that you won't be able to get this information from a 2nd gen system.

[Melissa]

Agree with westermann. I'm sure you know the location of that repeat. Just design primers flanking that region and amplify it. Forward and reverse sequencing using Sanger should do the job. After all, it's just 1kb!!!!

[pmiguel]

Agree with westermann. I'm sure you know the location of that repeat. Just design primers flanking that region and amplify it. Forward and reverse sequencing using Sanger should do the job. After all, it's just 1kb!!!!

Probably won't work. If these are identical short tandem repeats there is no basis to align the forward and reverse reads.

Phillip

[Claudia Stewart]

Has anyone seen a very high percentage of dots whenlooking at tandem repeats (80%)? Can you adjust the reaction to read through these and/or capture the data back from the filter to analyse what is really going on?