I have some primers that have been used successfully to amplify the V1-2 region of the bacterial 16S rRNA gene. I plan to run some 16S rRNA sequencing analysis and I must now add a sequencing adapter sequences to each of my primers respectively. Should I calculated the Tm based on the 16S-specific primer region only, or should I calculate the Tm with both parts of the primer (seq adapter + 16S specific region)? The addition of the sequencing adapter increases the size of the the primers and therefore the Tm significantly above recommended annealing temperatures.
Thanks in advance!
The following article is with respect to preparation of amplicon sequencing library.
A novel ultra high-throughput 16S rRNA gene amplicon sequencing library
preparation method for the Illumina HiSeq platform
(https://microbiomejournal.biomedcentral.com/track/pdf/10.1186/s40168-017-0279-1.pdf)
No idea what analysis you are goign to do!!!
"I plan to run some 16S rRNA sequencing analysis and I must now add a sequencing adapter sequences to each of my primers respectively."
You my get some useful information by reading the article given
Good Luck!!!
Hey!
According to Illumina 16S guide, for Tm calculation you need only target-specific region of primer.
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