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Old 08-13-2020, 02:21 PM   #1
techfromheck
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Exclamation Help with ideas for barcode screw up

Got any sequencing wizards out there? Undergrad barcoded 10x 96 well 16S amplicon plates for a miSeq muliplex run with the same set of Nextera barcodes for all the plates. Ideas other than starting over?
I'm wondering if I can ligate another barcode with a different adapter sequence for a hiSeq (like UD) so each plate has a unique barcode then each well would have an its x,y combination.
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Old 08-14-2020, 09:32 AM   #2
cmbetts
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You'd likely run into some nasty color balance issues just trying to add on an external adapter, and who knows what would happen during clustering with internal P5/P7.

Then you're especially stuck on a MiSeq. The cartridge premixes all of the sequencing primers, so you'd be reading out the gibberish combination of both two read primers simultaneously.

Best I could suggest is doing some extra amplification on your existing libraries with all new index sequences (enough that they'd become the majority of molecules, but not too much that you risk the relative abundance in the library). Then you can use only the new indexes in your sample sheet and the overlapping index reads will get sent to the unidentified reads fastq. You'd probably lose some sequencing capacity, but may still be worth it depending on the sample value and other concerns.
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