Paired end reads DNA strains relationship.

Question

[arronar]

Hello.

A) I just wondered about the relationship of two paired end DNA strains in NGS method. If we assume that we can read the whole DNA fragment, then the two DNA strains will be the same or they will be complementary of each other ?

B) Also which are the basic reasons that makes difficult to read big DNA fragments ? If I'm correct, I think that we can read strains shorter than 500bp.

Thank you

[GouthamAtla]

We don't read the whole DNA fragments. We break the DNA in to short fragments and read from both the ends to get a kind of long reads. If we read the entire DNA at a stretch (impossible for now), there is no need to sequence from both the ends.

Answer 1

[Stoploss25]

The commenter above was right but it also depends on your insert size. Your cDNA insert is what actually gets sequenced and if the length of it is less than 2 * readlength you will have some overlap or 'dovetailing' I think its called.

Some methods of sequencing can now go longer than 500bp. Pacific Biosciences has a sequencing technology that can give 6kb reads on average, with some up to 10kb. The tradeoff is it has a much higher error rate, and you get much less reads.