Greetings,
We have been getting low cluster densities, and as we increase the concentration, we are getting less clusters/tile/pM. It looks like there is no way for our samples to reach the optimal density of 400,000. It would seem that there is something wrong with our RNA-seq samples other than concentration that is causing this trend.
Ex:
9.6pM – 175,000 cl/tile (18,000 cl/tl/pM) – one lane
10pM – 146,000 cl/tile (15,000 cl/tl/pM) – one lane
20pM – 279,000 cl/tile (14,000 cl/tl/pM) – one lane
22pM – 261,000 cl/tile (12,000 cl/tl/pM) – one lane
25pM – 120,000 to 240,000 cl/tl (5,000 to 10,000 cl/tl/pM) – seven lanes, seven diff samples
We are multiplexing – 6 barcodes per lane. Samples were quantified with the Qubit flourometer. The bioanalyzer showed multiple peaks for our earlier samples that were daisy-chained. The extra peaks were eliminated when we changed the gel extraction protocol from 50C to RT, and the bioanalyzer showed very nice peaks at approx 350bp. Recently, one of our samples was checked with qPCR and found to be ¼ the concentration of the Qubit reading. It had previously been sequenced at 25pM and yielded 150,000 cl/tl.
1) Does anyone get such drastically different concentration readings?
2) Any ideas as to why so little of the sample is sequencing when the bioanalyzer shows our library is the target length in bp and PCR enrichment works well?
3) Any ideas as to why higher concentrations are not increasing (often decreasing) the cluster density?
I greatly appreciate any ideas or help you may have on these problems. Thanks, thanks and thanks!!
We have been getting low cluster densities, and as we increase the concentration, we are getting less clusters/tile/pM. It looks like there is no way for our samples to reach the optimal density of 400,000. It would seem that there is something wrong with our RNA-seq samples other than concentration that is causing this trend.
Ex:
9.6pM – 175,000 cl/tile (18,000 cl/tl/pM) – one lane
10pM – 146,000 cl/tile (15,000 cl/tl/pM) – one lane
20pM – 279,000 cl/tile (14,000 cl/tl/pM) – one lane
22pM – 261,000 cl/tile (12,000 cl/tl/pM) – one lane
25pM – 120,000 to 240,000 cl/tl (5,000 to 10,000 cl/tl/pM) – seven lanes, seven diff samples
We are multiplexing – 6 barcodes per lane. Samples were quantified with the Qubit flourometer. The bioanalyzer showed multiple peaks for our earlier samples that were daisy-chained. The extra peaks were eliminated when we changed the gel extraction protocol from 50C to RT, and the bioanalyzer showed very nice peaks at approx 350bp. Recently, one of our samples was checked with qPCR and found to be ¼ the concentration of the Qubit reading. It had previously been sequenced at 25pM and yielded 150,000 cl/tl.
1) Does anyone get such drastically different concentration readings?
2) Any ideas as to why so little of the sample is sequencing when the bioanalyzer shows our library is the target length in bp and PCR enrichment works well?
3) Any ideas as to why higher concentrations are not increasing (often decreasing) the cluster density?
I greatly appreciate any ideas or help you may have on these problems. Thanks, thanks and thanks!!
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