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How to get uniform data yields per barcode in nanopore sequencing

When we perform nanopore sequencing on DNA-barcoded pool of samples, we frequently see too much variability in data yields between barcodes (i.e. samples). In a run with 24 yeast samples on one flowcell, we typically see that the barcode with the least data has 2 to sometimes 10-fold lower data yields compared to the barcode with the most data. What are the best practices and recommendations to keep data yields uniform across barcodes within a pool?

ont nanopore
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updated by samuel.a.odonnell samuel.a.odonnell • written by Irsan Irsan

I have the same experience with many different runs and many different extractions, library preps etc. I think the throughput variation quite commonly comes down to differences in molarity between samples. So keeping a similar read length and concentration within all the samples helps, but with that being said that is rather difficult with the ligation kits (due to the bead washes). \ Also sometimes I have just had extractions that look like others but do not work very well...its hard to control the variability. \ Goodluck

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written by samuel.a.odonnell samuel.a.odonnell

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