Improving sequencing yield is a goal we all have. Tonight I watched the video from the Nanopore Learning course on Human genome sequencing and analysis with the title “Improving sequencing yields: Getting the most out of your samples.” They noted the importance of keeping the flow cell pores full. For this, they recommend 5-50 fmol of good quality library. For short fragments shorter than 10 kb, they recommend starting with 100-200 fmol to avoid the risk of overloading the flow cell. Short fragment libraries are easier to quantify the molarity. For long fragments greater than 10 kb such as unsheared gDNA, they recommend starting with 1 ug, since there is the risk of under-loading the flow cell. However, longer fragment libraries are harder to quantify the molarity. The video noted that for the ligation sequencing kit (LSK), <500 ng of gDNA may result in throughput drop. They recommend fragmentation to increase the number of “threadable ends.” For this, they recommend 100-500 ng of input. The disadvantage is that shearing will not enable ultra-long reads. For less than 100 ng, ONT suggests PCR to maintain throughput. For this, they recommend using the low-input PCR-based kits, for example. Clogs can be caused by contaminants getting into the pores between sequencing molecules of DNA. The rapid kits suggest 400 ng of DNA. Throughput is determined by the number of cuts, not input amount. Increasing the throughput can improve read length… even creating “monster read lengths.” Size selection can be accomplished using BluePippin systems, though ONT noted that you still get some small fragments. SPRI beads can be used as a “crude alternative to BluePippin.” Without size selection, the speaker noted that “a lot of time is spent sequencing short reads.” They also recommend visiting Protocols.io to learn about new protocols to increase yields. This video was helpful as it explained pore occupancy and library sizing!
