Thursday, May 6, 2010

Nextera™ library fragment size range

Another common question regarding our Nextera™DNA Sample Prep Kits for next-generation sequencing:

The Nextera library fragments are too small. Is there a better way to control the DNA fragment size?

Nextera DNA Sample Prep kits include two buffers, Low Molecular Weight (LMW) and High Molecular Weight (HMW), for generating two size classes. In general, with the Nextera Illumina-Compatible Kit, the LMW buffer will produce fragments from 150 to 600 bp (peak around 200 bp). The HMW buffer will produce fragments from 175 to 700 bp (peak around 250 bp). The final fragment size includes approximately 100 bp of adaptor/transposon end sequence, so the actual genomic fragment is smaller than the apparent MW of the library. It is normal for the fragment MW to vary slightly. This is commonly a result of DNA type, quality, and purification and quantitation methods used.

The quality of the starting DNA is critical. Contaminants such as protein and RNA may inhibit the Nextera tagmentation reaction if present in the DNA preparation. Therefore, it is important to start with highly pure  DNA. We also recommend using HMW buffer if small fragment size is an issue. The current protocol has been developed with 50 ng of DNA. If the fragments are too small, we recommend adding slightly more DNA. Changing the temperature or reaction time is not a robust or reproducible way to control fragment size. Finally, if a narrow MW distribution is required, it may be necessary to perform a size-selection step (gel, AMPure® beads, SPRIWorks®, etc.) after the limited-cycle PCR.

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