Hello!
Ive been trying to optimize a protocol to be able to sequence 6-8kb expression plasmids (like pET21 containing a variety of genes of interest) in a multiplexed barcoded manner using the flongle. My current workflow is Bacterial Colony -> Heat Lysis -> Rolling Circle Amplification using Phi29 -> Heat Inactivation -> Barcoding using SQK-RBK114.96 -> Pooling of samples -> adapter ligation and sequencing. After quite a bit of optimization, Ive been able to consistently get a large number of reads per barcode (5k-20k with most reads in the range of 0.5-1.5kbp) yet the assembly file is full of errors. There are either a lot of point mutations or big chunks of sequence missing. Im currently generating the assemblies using the standard Epi2Me "coding-free" software they provide for plasmid sequencing. Is this known to be a poor way to analyze sequencing data? Is there a way to pass in a reference sequence during the assembly process to improve the read assembly efficiency? Thanks so much!
Heres the workflow it seems to be running for the assembly https://github.com/epi2me-labs/wf-clone-validation
See if you are able to work with ONT support/local applications scientist. Sounds like you have some issue going on with your prep (hopefully not with sequencing assuming you have done sequencing before with other samples). While you may see a few reads with oddities it should not be a universal thing.
Is that supposed to selectively amplify just plasmid DNA?