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What factors affect whether I will see an improvement with adaptive sampling?

What factors affect whether I will see an improvement with adaptive sampling?

A few things will influence whether an adaptive sampling approach will be better than a non-adaptive sampling run.

  1. Adaptive sampling requires a considerable higher molarity in the sample than WGS. It is advised that ~ 50 femtomoles are used instead of the recommended 10 femtomoles for WGS sequencing.
  2. The read length of the sample can also affect enrichment in 3 ways:
  • Firstly it will play into the molarity of the sample (if read length is too long, then preparing a high molarity sample can be complicated).
  • Long reads will increase the blocking rate of the flow cell (this effect is more serious in AS runs when compared to WGS - ideal library range between 6-15kb).
  • If your targets are small (i.e. 2-3 kb) and your library is long (i.e. 15 kb), every time a strand is accepted for sequencing only 2-3 kb out of 15 kb will be useful, and you are wasting sequencing time and pore vitality, hence it is not as advantageous.
  1. The amount of the sample targeted. Your enrichment potential is inversely proportional to the percentage of the sample targeted. Ideal targeting ranges are between 0-5% of the sample with a recommended maximum at 10%.
  2. Load on the machine can influence the speed with which MinKNOW can make a decision on the captured strands and this will play into the final enrichment as well. In particular for PromethION devices, a large number of flow cells performing Live basecalling and/or alignment can influence the amount of resources available to quickly make Adaptive sampling decisions.

More information about adaptive sampling can be found in the guide here.

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