Queries on 10x sequencing 3' Read 1
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7 weeks ago
Cheng Wei • 0

I have seen on umi_tools that the read 1 only contains the cell barcode and UMI. However, when I look on my data, the read length is much longer than 28. Does that still mean that my read 1 contains only the cell barcode and UMI?

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Yes, the rest might be adapters or garbage, you can blast it if you like.

Here is an absolutely excellent resource on single cell lib structures.

https://github.com/Teichlab/scg_lib_structs

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The rest of R1 would be the oligo dT primer and eventually the 3' sequence of the transcript. Illumina sequencing would have a hard time with base calling on the polyA region though due to the low complexity problem.

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That having said, it is on the user to define on the sequencer how long a read is. If you sequence the library as a 2x150bp run (very common run mode on Novaseq machines, most cost effective) then R1 is 150bp, but the meaningful part is much shorter as said above. One would simply discard (many tools do that automatically) everything after the CB/UMI part as it is basically garbage.

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