-
Below is a list of the most commonly encountered issues, with some suggested causes and solutions.
We also have an FAQ section available on the Nanopore Community Support section.
If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.
-
Troubleshooting
Observation Comments and actions Low throughput 1. Vortex gently after adding the diluted Fragmentation Mix (FRA) to break up the largest fragments.
2. Ensure the diluted Fragmentation Mix (FRA) is thoroughly mixed with the gDNA.
3. Use less input material if the DNA library was too viscous to load onto the flow cell.DNA is too viscous and will not load onto a flow cell 1. Lower the input material to reduce the amount of gDNA going into the library preparation and reduce viscosity.
2. If DNA library will not load using the method outlined in this protocol, slowly pipette mix 5 times with a standard P200 pipette set to the full volume of the library and reload the flow cell.Read lengths are not long enough 1. Increase input material.
Note: Library viscosity increases with more gDNA.
2. Reduce volume of Fragmentation Mix (FRA) added to FRA Dilution Buffer (FDB) to avoid over-fragmentation of gDNA.
Note: We do not recommend diluting less than 2 µl Fragmentation Mix (FRA).
3. We recommend using PFGE to check the extracted gDNA is of ultra-high molecular weight (uHMW), thus capable of generating ultra-long read lengths.No sequencing output 1. Check gDNA has been recovered in library preparation using a NanoDrop spectrophotometer.
2. Check viscosity of the sample. The library should be viscous if it contains uHMW gDNA in this protocol.Aspirating supernatant when the DNA has precipitated Take care to not aspirate the DNA. Remove smaller volumes of supernatant incrementally to reduce the risk of aspirating the DNA. Mixing Mix slowly and carefully to prevent DNA shearing. Low vortexing can be used to mix at the expense of ultra-long reads. With vortexing, long read lengths of ~90 kb N50 can still be generated with improved outputs. No DNA recovered from the library preparation clean-up If the DNA is no longer viscous or the NanoDrop reading is low, DNA may have been lost during the clean-up step of the library preparation.
1. Ensure uHMW DNA is used or users risk DNA loss.
2. Take care to not aspirate the precipitated DNA during the clean-up step. To avoid this, remove smaller volumes of supernatant incrementally. Ensure as much supernatant is removed as possible.