I am looking for suggestions to improve the yield of my libraries prepared using Illumina True seq small RNA library prep kit. Input RNA is purified small RNA (50 ng) and I run for 15 PCR cycles. I use gel runs for size selection because these kits create a lot of adpater dimers.
Final library yield is coming around 10 ng, which is unacceptable for sequencing. What's the best way out to this problem?
Hi Mansi, I have encountered somewhat similar situation on AmpliSeq Ion torrent platform. Personally I won't recommend any drop in PCR cycles to avoid the notorious adapter dimers, as this will definitely contribute in low yield of library, especially in certain cases where the input template quality is not very good like in FFPE or in fragmented uneven-sized template cases, for example; performing the reverse transcription step based on the priming strategy containing only the random hexamers and no Oligo dTs at all. So we have bundles and bundles of tiny fragments far less in sizes than the required sequencing length.
Insufficient starting template quantity also results in adapter dimers. Try to increase the template input more. Increase few more PCR cycles as allowed in your protocol. Insufficient cleanup of libraries also result in adapter dimers and low library yield so focus much on cleaning & purification steps (I always prefer to optimize this step, not just obey the protocol).
If still the adapter dimers making you nervous, just use diluted one or increase their dilution or even you can use lesser volume of your adapters than the recommended one.
You need to concentrate quite hard in the cleaning and purification step, as any residual ethanol or any other solvent eventually inhibits or slows the PCR, which also result in low or negligible library output. And more often the major problem lies in this step, which is overlooked by newbies.
Regards...
Jawwad Ahmad Thanks for your suggestion. I defenitely do not want to increase my PCR cycle numbers as it will result in an increase in adapter dimers.
Cleanup is the most crucial step of library preparation and choice of cleanup methods determines the final yield. In my case, I use gels to effeciently distinguish adapter dimers with libraries as in my case these are very close in size. I am trying to increase the input amount which is quite challenging for purified small RNA.
Thanks a lot !
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