Using the rapid sequencing kit for cDNA

jgibbons1 · 11-19-2021, 10:51 AM

We haven't done RNA-seq with the minion yet, but have an extra Rapid Sequencing kit lying around. I am wondering if there's anything "wrong" with creating our own cDNA and then sequencing it with the Rapid Kit, rather than using the one of the cDNA Sequencing Kits.

We are interested in some isoforms between two samples and thought this might be a creative solution with the kit we have in hand.

Any suggestions would be appreciated!

Thanks.

jgibbons1 · 11-29-2021, 05:12 AM

To answer my own question, after thinking about this more...

This kit includes a fragmentation step, which would result in cDNA fragmentation and limit sequencing cDNA of full in tact isoforms. There's a risk the isoforms we're interested in will be fragmented.

gringer · 01-18-2022, 06:23 PM

Also, the cDNA kits are a stranded sequencing process (different adapter sequences for forward and reverse); you'd lose that benefit by using either the ligation kit or rapid kit on pre-prepared cDNA.

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11-19-2021, 10:51 AM	#1
jgibbons1 Senior Member Location: Worcester, MA Join Date: Oct 2009 Posts: 135	Using the rapid sequencing kit for cDNA We haven't done RNA-seq with the minion yet, but have an extra Rapid Sequencing kit lying around. I am wondering if there's anything "wrong" with creating our own cDNA and then sequencing it with the Rapid Kit, rather than using the one of the cDNA Sequencing Kits. We are interested in some isoforms between two samples and thought this might be a creative solution with the kit we have in hand. Any suggestions would be appreciated! Thanks.

11-29-2021, 05:12 AM	#2
jgibbons1 Senior Member Location: Worcester, MA Join Date: Oct 2009 Posts: 135	To answer my own question, after thinking about this more... This kit includes a fragmentation step, which would result in cDNA fragmentation and limit sequencing cDNA of full in tact isoforms. There's a risk the isoforms we're interested in will be fragmented.

01-18-2022, 06:23 PM	#3
gringer David Eccles (gringer) Location: Wellington, New Zealand Join Date: May 2011 Posts: 843	Also, the cDNA kits are a stranded sequencing process (different adapter sequences for forward and reverse); you'd lose that benefit by using either the ligation kit or rapid kit on pre-prepared cDNA.