Hi Everyone,
Although I have been visiting the site for quite some time, this is my first time posting on seqanswers. To get down to it, I have a specific question about using Truseq indexing primers for differential miRNA expression analysis on the Hiseq 2000 machine. As I understand it indexing primers can cause differences in amplification efficiency for different sequences within a library and these differences will distort the library in different ways depending on the indexing primer. So, the end result would be that one cannot accurately compare samples from different conditions that use different indexing primers (I hope that's not too convoluted). I could be wrong about this as perhaps illumina has done something to alleviate bias generated through amplification with differing indexing primers. I guess what I'm wondering: is this a legitimate concern? If so would swapping indexing primers between conditions from one run to the next alleviate this problem?
Thanks in advance,
Graduate Student
Although I have been visiting the site for quite some time, this is my first time posting on seqanswers. To get down to it, I have a specific question about using Truseq indexing primers for differential miRNA expression analysis on the Hiseq 2000 machine. As I understand it indexing primers can cause differences in amplification efficiency for different sequences within a library and these differences will distort the library in different ways depending on the indexing primer. So, the end result would be that one cannot accurately compare samples from different conditions that use different indexing primers (I hope that's not too convoluted). I could be wrong about this as perhaps illumina has done something to alleviate bias generated through amplification with differing indexing primers. I guess what I'm wondering: is this a legitimate concern? If so would swapping indexing primers between conditions from one run to the next alleviate this problem?
Thanks in advance,
Graduate Student