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1 hour ago by

While the R1 read is anchored at the poly-A tail, the R1 read contains mostly the various barcodes and UMI that are part of the adapter. The reads really being aligned to the genome are the R2 reads, which can be some distance from the poly-A tail depending on the fragment size of the insert.

As for the intronic reads you see, due to imperfect priming in reverse transcription, roughly 10-30% of reads are primed within gene introns due to incidental poly-A runs in the genome. There is actually a computational cell trajectory method that takes advantage of this called RNA velocity, because intronic reads are mostly derived from nascent transcripts.

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