Typical Quantity of Primer Sequences In Final Library

[Dario1984]

Hello,

I'm wondering what amount of contamination is typical of a multiplexed library ? We've just done our first ever multiplexing run through a service provider on the HiSeq for a number of ChIP-seq experiments against histone mark H2A.Z. Here is FASTQC output for one sample :

Basic Statistics
Total Sequences 35111957
Sequence length 51

Overrepresented sequences
No code has to be inserted here.Also, are there any protocol tricks so that tags aren't being wasted just sequencing primers ?

I realise there's also the problem of overamplification here (3.5 million unique mapping reads with Bowtie). I hear that happens often when indexes are used ?

[jlove]

Dario,
It sounds like you're doing ChIP Seq... are you using the TruSeq kit? If so, I can tell you what we do. I use 10 to no more than 50 ng of input as measured by pico green, and a 1:50 dilution of the adapters. Usually 15-18 cycles of PCR are required. I also do the size selection AFTER the PCR, which removes any dimer.