Did not take note of my indices (barcodes); is my library salvageable?
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28 days ago

Hi everyone. I have a rather annoying problem:

I planned to sequence two small RNA libraries I created a few months ago.

I neglected to write down the indices that I used to create these libraries, and now I am at a loss as to how to continue. Is it possible to perform PCR to detect my barcodes? Can I run these libraries without knowing what the barcodes are right now, and demultiplex later? Or do I need to start over?

RNA index iSeq sequencing • 598 views
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If the libraries haven't been pooled yet you could sanger sequence the index for each sample.

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Like a PCR with p5 and p7 primers? I have not pooled yet, but I have libraries with relatively wide distribution of sizes...would that be a problem for sanger?

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I don't know what adapters you are using, so you'll have to see if p5 and/or p7 are sufficient for sanger sequencing, or if you need to design your own primers.

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I would use p5 and p7...but is it okay for sanger that I have a distribution of amplicons (130-180bp)?

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After all you can also first amplify an aliquot per library (normal amplification PCR), put on gel, excise a precise band and then send that for Sanger to have a defined product size. It does not matter since the barcode is the same regardless of amplicon length. That should be enough to find out the barcode. Learn from it, library prep documentation is so important!

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27 days ago

If you know which tube is which, you could run them separately, and figure out what the barcode must be by looking at the barcodes of undetermined reads.

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I guess someone is being yelled at if the bill for one sample per lane comes in…

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It might not be that bad on a miseq. Or, it can share a lane, (if you know something about what it might be, and know it won't conflict with the known barcodes there)

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