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Hello,
I'm preparing libraries with Nextera XT. From 40 samples 7 are not alright. They are shorter than the rest of my libraries and I'm unsure about sequencing results from these samples. I would like them a bit longer (peak at 300+bp) to ensure the sequencing is successful. Right now I'm using 4 min tagmentation (the samples contain preamplified genomes) which generally gives me good results but these few samples are problematic. I was thinking of reducing the tagmentation time by half but I'm not sure it is long enough to work for sequencing. I tried adding more DNA without change. I'm attaching two samples on Agilent, one is definite no and the other could be longer (3 samples have peaks under 200bp, 4 under 300bp). I might split the samples and try 3 min and 2 minutes tagmentation? Can anyone help? Is it possible to have this short tagemntation time and still insert all needed sequences? Any other advice about modifying the protocol to gain longer fragments?
Thank you so much for your help,
Dominika
I'm preparing libraries with Nextera XT. From 40 samples 7 are not alright. They are shorter than the rest of my libraries and I'm unsure about sequencing results from these samples. I would like them a bit longer (peak at 300+bp) to ensure the sequencing is successful. Right now I'm using 4 min tagmentation (the samples contain preamplified genomes) which generally gives me good results but these few samples are problematic. I was thinking of reducing the tagmentation time by half but I'm not sure it is long enough to work for sequencing. I tried adding more DNA without change. I'm attaching two samples on Agilent, one is definite no and the other could be longer (3 samples have peaks under 200bp, 4 under 300bp). I might split the samples and try 3 min and 2 minutes tagmentation? Can anyone help? Is it possible to have this short tagemntation time and still insert all needed sequences? Any other advice about modifying the protocol to gain longer fragments?
Thank you so much for your help,
Dominika