Illumina PE 100bp and allele content

[yog77]

Will all of your amplicons be exactly 200 bp long?
And are you aligning to a reference genome after sequencing?
I'm just thinking that if you're not aligning, and your reads don't overlap, then how will you know the actual insert size -- i.e. how many nt are between your PE reads.

In terms of size, though, having some be less than 200 nt and having the PE reads overlap is no problem. I've seen a number of our samples have low insert sizes close to a mean of 180 nt, with a distribution around that (i.e. some < 150 nt), and aligning to the reference human genome has been no problem.
You just don't want your inserts to be so short that you start reading through them into the adaptor sequences. But it sounds like that wouldn't be the case here.

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Thanks for the quick response

I was hoping to generate similar sized amplicons (~200bp) so there would be no need for size selection.

I plan to do bisulphite sequencing and aligning to a small region where all my amplicons will come from (300kb region of bisulphite converted sequnce) some of these amplicons will overlap with one another.

In essence I want as much read length (100bp x2) from the 200bp PCR amplicons as possible and so I was thinking there was going to be no insert - is this possible?

[Jeremy37]

Will all of your amplicons be exactly 200 bp long?
And are you aligning to a reference genome after sequencing?
I'm just thinking that if you're not aligning, and your reads don't overlap, then how will you know the actual insert size -- i.e. how many nt are between your PE reads.

In terms of size, though, having some be less than 200 nt and having the PE reads overlap is no problem. I've seen a number of our samples have low insert sizes close to a mean of 180 nt, with a distribution around that (i.e. some < 150 nt), and aligning to the reference human genome has been no problem.
You just don't want your inserts to be so short that you start reading through them into the adaptor sequences. But it sounds like that wouldn't be the case here.

[yog77]

Hi All Im new on here. i was after advice concerning the 100bp PE reads:

Q1) I have heard it is a problem bioinformaticaly that if you do 100bp PE reads, you ideally don't want the reads from either end to overlap (ie more than 100bp+ from either end) coz if they do you can't align these easily.

Q2) Following on from Q1 the reason I ask this is that I would like to sequence directly from 200bp PCR fragments as I am hoping to index 96 samples in a single lane. So is it possible to sequence many (~150) amplicons that are all 200bp long, I guess adapters and barcodes would be added to these after PCR.

so ~150 x 200bp amplicons for 96 patients using 100bp PE reads, therefore achieving 200bp reads that would hold allele specific information for that one paired end read. Also I really have no other option but to use PCR products - but to maintain the read along the full length.

Can anyone help