We have a theory that large libraries, specifically those with fragments trailing off to >2kb, do not quantitate accurately from Bioanalzyer. We think this because qPCR concentrations do not correlate. Has anyone else had this problem? It seems if we are able to remove the "tail", then qPCR matches a bit better with Bioanalzyer.
-
-
Libraries over 2 kb don't tend to work on Illumina sequencers, so do you really care what the concentration of those molecules is?
Also, are you doing a TaqMan qPCR or SYBR? If it's SYBR, you have to factor in the average molecule size, so you're still dependent on the Bioanalyzer anyway. -
This depend on qPCR cycle settings. Using standard protocols gives enough time for extension of around up to 900 bp fragments so larger fragments are not amplified by qPCR. Since fragments above this size do not cluster efficiently they can be ignored. For accurate quantification average fragment length from the shortest fragment to 900-950 bp on the bioanalyser can be used to calculate concentration.Originally posted by kgoglin View PostComment
-
Researchers using sequencing and genomics tools often have to make trade-offs. They can choose between speed or scale, short reads or long-range information, or targeted panels or a view of the whole transcriptome. New technologies that have been released this year are built to address those tough choices.
We asked six companies the same four questions to learn about their latest products. The new technologies bring a lot to the table, including rethinking sequencing...
Comment