We get a number of inquiries about whether the Nextera™ Read 1, Read 2, and Index Read sequencing primers are compatible with the standard Illumina® sequencing primers. The Nextera and Illumina sequencing primers are indeed compatible with each other. The sequences of Nextera primers are different from standard Illumina primers, which means that they will not interfere with or cross-hybridize to each other. Nextera primers also will not anneal to a library created using standard Illumina sample prep technology and vice versa. This means that it is possible to sequence both libraries, not only in the same flow cell, but also in the same channel, as long as the libraries are suitably barcoded.
For libraries prepared using Illumina TruSeq™ technology: as with the standard Illumina libraries, Nextera libraries can also be sequenced in the same channel of the same flow cell, again due to the differences between Nextera primer and TruSeq primer sequences. Thus, with appropriate barcoding, it is possible to sequence a Tru-Seq library, a standard Illumina-generated library, and a Nextera library in the same flow cell, without fear of primer interferences or sequence data confusion.