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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.

  • Adaptive sampling requires a higher molarity in the sample than WGS. It is advised that ~65 fmol of library are loaded instead of the recommended 50 fmol for whole-genome sequencing.
  • The fragment length of the sample can also affect enrichment in three ways:
  1. Firstly, it will play into the molarity of the sample (if read length is too long, then preparing a high molarity sample can be challenging).
  2. 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).
  3. 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, using sequencing time and pore vitality inefficiently.
  • 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%.
  • 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 Adaptive sampling guide.

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