The beginning of the third day we will enrich the library, initialize the PGM, load the chip and we’re off and running. I don’t want to push people or throw too much at someone when they are starting next generation sequencing, because it’s a lot just to figure out how to use your time most efficiently, but it really is very smooth to get to that library quantification and start the template prep which will go over night on the Ion One Touch™ instrument. Sometimes we start the template preparation that same day, which I think is great. Adding too little DNA will reduce the number of reads obtained for the sample because there will be more 'bald' or untemplated beads (Ion Sphere™ Particles) there is unused real estate on the chip. Adding too much DNA to the emulsion PCR increases the polyclonal reads, which are not interpretable by the instrument and are discarded. This is important because the ratio of DNA to Ion Sphere™ Particles (or beads) for the template preparation will dictate whether the emulsion PCR is clonal (and can be sequenced on the PGM) or polyclonal. And I should mention too, after library preparation, performing an additional library quantification is key. We like to get all of the library prep, including a whole series of different incubations in the thermocycler, done in one day so that by the end of the day we can quantify the library. It usually takes a full day to macro dissect, extract on the Maxwell® instrument, and quantify before we are ready to do library preparation the following day.
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