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mritchielab / restrander

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10x - 3' kit primers #5

Open sparthib opened 2 months ago

sparthib commented 2 months ago

Hi there, I was wondering what the difference between RTP/ VN and a forward primer is based on the 10x - 3' protocol doc. The TSO in my protocol seems to match the one in the restrander config file. However, it isn't clear to me what the "VN" in my kit is. However, the forward primer introduced in the cDNA amplification step of my protocol has the same sequence as the RTP in the config.

The amplified reads end up looking like this:

image

If you have any clarification on this, it would be great! Thanks!

Sowmya

QGouil commented 2 months ago

Hi Sowmya, we took the VNP/RTP terminology from the ONT protocols, but as you’ve correctly identified, it’s the "forward" primer sequence in the 10x protocol. "Forward" and "reverse" are ambiguous depending on which way you draw the amplicon, but the TSO/SSP needs to be the sequence that’s introduced near the transcript start, and the VNP/RTP/"forward" the sequence that’s introduced near the transcript end (towards the polyA). I hope this clarifies things!