Monday, March 1, 2010

Tangled fragments are “for the birds”: Bird-nesting explained

Why do the fragment sizes in my Nextera™ library seem longer than the 200-400 bases expected?
We’ve had a few customers ask this question when using the Nextera DNA Sample Prep Kit (Illumina-Compatible). 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.

Illumina-compatible sequencing libraries were prepared using Nextera LMW buffer according to the standard protocol. PCR products were purified using either Zymo (red trace) or AMPure beads (blue trace) per the manufacturer’s instructions. The resulting sequencing libraries were examined using a BioAnalyzer (Agilent). The traces show an example of “bird nesting” where noncovalent concatamers result in higher apparent molecular weight under native conditions.

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