Wednesday, March 16, 2011

Preparing Nextera libraries from short linear DNA

Occasionally, we get requests for information on the use of the Nextera™ DNA Sample Prep products for sequencing short linear DNA, such as PCR products or cDNA libraries. Nextera kits can be used with these targets, though a minor change in the procedure will be required.

Due to the mechanism of DNA “tagmentation” by Nextera Transposomes, the end sequences (the last 100-150 bases) of the target DNA will have lower depth of coverage than the center of the DNA. This is because the Transposomes are not capable of adding adaptors to the ends of the DNA, and because at least two Nextera transposon insertions are required to tagment a given DNA molecule. A relatively simple fix is to design PCR or cDNA primers that include one of the Nextera Adaptor 1 and Adaptor 2 sequences; alternatively, ligate the adaptor sequences to the ends of the amplicon or ds cDNA. This will allow the end sequences to participate in the limited-cycle PCR after tagmentation, and allow for improved coverage of the ends of the DNA.

Another option when using PCR amplicons is to design PCR primers so that the priming sites are outside the desired region for sequencing. This will increase the likelihood that a Nextera Transposome complex will insert much closer or next to the end of the sequence being studied.

2 comments:

Vitaly Korchagin said...

Can we use Nextera Sample prep. kit to produce the library from PCR-products mix with 90-150 bp size range?

epibio said...

That size range would be too small to get a good representation Nextera library. Also, you should be aware that this version of the Nextera kit has been discontinued. The current version is sold by Illumina.