Sequencing NEXTERA library with high average

[Mario Moreno]

Hi,
We have one question,
We tried using nextera for fragmentation of bacterial DNA genomic , but this non fragmented completely.
We change the concentrations of DNA initial, time of fragmentation, with kit NEBNext, sonication, and for heat ,but nothing...
The best result was one library with average of 1368 pb (with nextera), we are thinking use this samples with a kit of 500 cycles, but we don´t know if this will result correctly.
Help me please!!!
Thanks!

[nucacidhunter]

Library looks a bit under tagmented but it is not an issue (if input DNA passed Nextera QC and was high molecular weight). If you use 20% less input DNA, (for this DNA sample) it should give a library with smaller average. I assume the trace is for library after PCR without bead clean-up. If that library is cleaned with 0.5x beads, it can be sequenced successfully. Without proper cleaning all those smaller fragments will be sequenced. The challenge will be quantification and getting optimum cluster density.

[snetmcom]

What did you try for sonication? I agree that lower input is what you should adjust for Nextera.