adapter contamination in reads?????

[meghavarshney]

hello everyone,

I would like to ask one qus: why am I getting 3'adapter sequences in R1 and R2 reads (as according to seq protocol machine only generates seq of the fragment between the adapters and not the flanking adapters)??
Also, is it normal to encounter abundance of 3'adapters in the reads than 5'. if so, why?

[dpryan]

Adapter read through with happen any time your read length is greater than the insert size. 5' contamination happens too, but more rarely.

[meghavarshney]

thanks for your reply , but i need to know about the main reason / logic behind this. And the other thing why am I getting 3' adapter seq in both reads( R1 and R2)???

[dpryan]

I already told you the main reason. Any time your read length is greater than the insert size you're going to sequence some adapters. These sequences will then be 3'.

[Michael.Ante]

The 3' adapter of R1 is the reverse complement of R2's 5' adapter and vice versa. Since you sequence and read in 5' -> 3' direction, you'll sequence into the 3' adapter any time your read length is greater than the insert size (see Devon's post above). Starting with the 5' adapter is very rare to nearly impossible. If I saw a 5' adapter contamination in Illumina libraries, I would assume that something in the lab went terribly wrong.

[meghavarshney]

Mr. Michael.Ante and Mr. Devon Ryan thanks a lot for sharing this information.