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-   -   barcoded RNA adapter ligation for multiplexing (http://seqanswers.com/forums/showthread.php?t=87662)

Lechu 02-13-2019 07:59 AM

barcoded RNA adapter ligation for multiplexing
 
Hi All,

This is my first post here, so hello everyone! :)

I'm wondering if it is possible to use RNA adapter ligated to (preferably) 3' end of the RNA for multiplexing the samples prior to RNA sequencing? If I understand correctly (an my knowledge may be narrow here), typically such adapters are added separately to individual samples, and then the barcodes are added later by PCR.

I would like to:

Ligate RNA adapters into multiple samples, and then pool them together before performing cDNA synthesis. After ligation, samples would be multiplexed, and processed as such from the very beginning. Specifically, I would like to perform a 3'UTR (or its polyA adjacent part) sequencing, using the custom adapter-specific primer containing barcode. My cDNA library would be made as follows, (with optional polyA enrichment):

........................................................................................... <-----------
....NNNNNNNNNNNNNN-AAAAAAAA [...] AAAAAA-BARCODE-ADAPTER

I would then use a random primer and adapter-specific primer for generating a second cDNA strand, and add adapters for amplification on the flow cell by PCR.

I can imagine such approach might lead to biases in case the RNA concentrations and/or quality in individual samples is highly variable. But assuming it is not, I see it as a sound approach. Or am I missing something?

Thank is advance for any helpful insights.

Cheers,
Lech

cmbetts 02-14-2019 12:52 PM

Very doable, although random priming isn't necessary for second strand if you're using standard Gubler-Hoffman, the RNaseH handles the priming. This Nature Methods paper from the Broad should give some inspiration on how to do it https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712044/


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