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.
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