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Hi,
I work on Miseq and I have some problems with the Nextera technology sequenced on Miseq.
I have started with Haloplex technology for bank preparation. I have determined a dilution factor for the injection on cartridge to generate 6.5 G of data with about 900K/mm² of clusters. We have used the same dilution for RNA-seq and the cluster generation was the same (850K/mm²).
For 2 weeks, I have been working with Nextera Enrichment with my own design. I have applied the same protocol as for Haloplex technology (dilution and injected quantity) for the library injection on the cartridge. Nevertheless, I have obtained a low number of clusters (100). I have changed NaOH solution and the cluster number has increased up to 300. Next, I have increased by 2 the quantity of library injection on the cartridge (24pM on cartridge instead of 12pM) but my cluster number has been about 500.
I have tested my DNA quality by qPCR and the result was good. Besides, the quantity assessed by qPCR was in accordance with my previous Qubit quantification as well.
Does anyone have any idea about my problem?
Is it a problem in my library generation or do I have to increase again and again the quantity of injected library ?
Thank you in advance for your precious help.
Sandy
I work on Miseq and I have some problems with the Nextera technology sequenced on Miseq.
I have started with Haloplex technology for bank preparation. I have determined a dilution factor for the injection on cartridge to generate 6.5 G of data with about 900K/mm² of clusters. We have used the same dilution for RNA-seq and the cluster generation was the same (850K/mm²).
For 2 weeks, I have been working with Nextera Enrichment with my own design. I have applied the same protocol as for Haloplex technology (dilution and injected quantity) for the library injection on the cartridge. Nevertheless, I have obtained a low number of clusters (100). I have changed NaOH solution and the cluster number has increased up to 300. Next, I have increased by 2 the quantity of library injection on the cartridge (24pM on cartridge instead of 12pM) but my cluster number has been about 500.
I have tested my DNA quality by qPCR and the result was good. Besides, the quantity assessed by qPCR was in accordance with my previous Qubit quantification as well.
Does anyone have any idea about my problem?
Is it a problem in my library generation or do I have to increase again and again the quantity of injected library ?
Thank you in advance for your precious help.
Sandy