illumina DNA library preparation, how to optimize the results?

[zhanghomer]

we used the adapter with a 3-bases tag in the 3' terminator.

The PCR primers are the universe primers from illumina, 16 cycles

the PCR products are tested by Agilent 2100, the results are below attachment.

as you see, noncovalent concatamerization of fragments (“bird-nesting”) was observed. so, how to optimize the conditions to reduce the bird-nesting.

[NextGenSeq]

Use the NuGen kit.

[andibody]

we too see it happen from time to time but we haven't been able to nail it down to specific conditions. hope we get more input from other users.

[zhanghomer]

Someone used another size selected gel extration followed the PCR amplification to solve this problem, but i think it will cut down the library quantity.

[andibody]

Nextera says bird-nesting isn't a problem:
"After the Nextera tagmentation reaction and PCR, you may notice an apparent fragment size of 500 bases (or greater) when the DNA is analyzed on an Agilent Bioanalyzer. This phenomenon is called “bird nesting”: the amplified, Transposome™-tagged DNA fragments get intertwined up and appear longer than they really are. This is not a problem. The denaturation step during bridge PCR will eliminate this tangle and the average peak size will settle back down to the expected 200- to 400-base range."

[peromhc]

I bet too much template DNA added to the PCR. Was it qualtified with Pico Green prior to the PCR step?

[andibody]

peaks of bianalyzer result seem small, wouldn't you expects lots of material if template DNA was abundant?
(I suppose 1 ul was used for quantification)

[zhanghomer]

I bet too much template DNA added to the PCR. Was it qualtified with Pico Green prior to the PCR step?

We qualtified the template DNA with Qbit, and add 50ng DNA template into each 50μl PCR reaction.

ps, the bird-nesting was appeared within 100 to 200bp, rather than "intertwined up and appear longer " , as Nextera says.

by the way, did anyone use the adapters with 3' index tag? do the tag index

will interfere the PCR efficiency ?