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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 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.
- 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.
- 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 guide here.
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