Hi everyone!
I'm currently doing a project in which I PCR amplify (long-range PCR) 20 genomic fragments (each of 5-10 kb in length) in 50 individuals. The aim is then to pool fragments of the same individual and mark each such 'pool' with a unique barcode. After doing so, the 50 individually barcoded pools will then be together sequenced on a single Illumina lane.
I'm aware that the best way to quantify PCR products for subsequent balanced pooling would be based on a Bioanalyzer measurement. Nevertheless, this would be too expensive and time consuming given that there would need to be done about 1000 measurements (50 x 20). Another way would be to gel-elute the PCR products and then measure the concentration (e.g. wih a Qubit). But again, this would lead to a total of about 1000 gel elutions...
As the PCRs are not working with the same fidelity I'm looking for a feasible way on how to quantify PCR products to pool them at more or less equal amounts for subsequent barcoding. The balancing doesn't need to be perfect, as I expect a very high coverage due to massive sequencing with Illumina (expected average coverage will be several 100x). Do you have any idea how I could efficiently quantify my PCR products for pooling? (I thought about putting each product on a gel and pool different amounts of respective PCR products based on the brightness of the band on the gel. I'm not sure, however, whether this is accurate enough).
I'm happy for any suggestions!
I'm currently doing a project in which I PCR amplify (long-range PCR) 20 genomic fragments (each of 5-10 kb in length) in 50 individuals. The aim is then to pool fragments of the same individual and mark each such 'pool' with a unique barcode. After doing so, the 50 individually barcoded pools will then be together sequenced on a single Illumina lane.
I'm aware that the best way to quantify PCR products for subsequent balanced pooling would be based on a Bioanalyzer measurement. Nevertheless, this would be too expensive and time consuming given that there would need to be done about 1000 measurements (50 x 20). Another way would be to gel-elute the PCR products and then measure the concentration (e.g. wih a Qubit). But again, this would lead to a total of about 1000 gel elutions...
As the PCRs are not working with the same fidelity I'm looking for a feasible way on how to quantify PCR products to pool them at more or less equal amounts for subsequent barcoding. The balancing doesn't need to be perfect, as I expect a very high coverage due to massive sequencing with Illumina (expected average coverage will be several 100x). Do you have any idea how I could efficiently quantify my PCR products for pooling? (I thought about putting each product on a gel and pool different amounts of respective PCR products based on the brightness of the band on the gel. I'm not sure, however, whether this is accurate enough).
I'm happy for any suggestions!
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