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Illumina DNA Prep/Nextera Flex with on FFPE samples, anyone?
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+ Ngs library preparation
+ Sample preparation
+ Next generation sequencing
+ Illumina
+ Sequencing
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Michelle Gibson
Illumina DNA Prep/Nextera Flex with on FFPE samples, anyone?
Muhammad Shakeel - thanks for the suggestion, I didn't think about reducing the tagmentation time. I thought that this parameter was significant with first version of Nextera, and that in Flex/DNA prep it did not matter. I'm afraid that I might be forced to look for end-repair method instead of nextera, which is so convenient. But the quality of input material is something that I have no control of. I also considered the FFPE repair kits - is it worth a shot? There were publications proving that it greatly improves the results of microarray analyses. I'm not sure if it will do the job in case of NGS.
Muhammad Shakeel - thanks for the suggestion, I didn't think about reducing the tagmentation time. I thought that this parameter was significant with first version of Nextera, and that in Flex/DNA prep it did not matter. I'm afraid that I might be forced to look for end-repair method instead of nextera, which is so convenient. But the quality of input material is something that I have no control of. I also considered the FFPE repair kits - is it worth a shot? There were publications proving that it greatly improves the results of microarray analyses. I'm not sure if it will do the job in case of NGS.
The major challenge in working on FFPE extracted DNA is the low quality (and quantity) of the input DNA. Please make double sure that you are using sufficient amount of input DNA. If your genomic DNA is degraded, yet the average size is >500 bp on an agarose gel, then you can proceed with the same library prep kit you are using. *But you will have to reduce tagmentation time!!* On the other hand, if your genomic DNA is highly degraded, my experience suggests not to use this kit, rather use the kit which follows end-rapair DNA --> adding of dA-tail ---> adaptors ligation workflow.
The major challenge in working on FFPE extracted DNA is the low quality (and quantity) of the input DNA. Please make double sure that you are using sufficient amount of input DNA. If your genomic DNA is degraded, yet the average size is >500 bp on an agarose gel, then you can proceed with the same library prep kit you are using. *But you will have to reduce tagmentation time!!* On the other hand, if your genomic DNA is highly degraded, my experience suggests not to use this kit, rather use the kit which follows end-rapair DNA --> adding of dA-tail ---> adaptors ligation workflow.
Muhammad Shakeel - thanks for the suggestion, I didn't think about reducing the tagmentation time. I thought that this parameter was significant with first version of Nextera, and that in Flex/DNA prep it did not matter.
I'm afraid that I might be forced to look for end-repair method instead of nextera, which is so convenient. But the quality of input material is something that I have no control of.
I also considered the FFPE repair kits - is it worth a shot? There were publications proving that it greatly improves the results of microarray analyses. I'm not sure if it will do the job in case of NGS.
Muhammad Shakeel - thanks for the suggestion, I didn't think about reducing the tagmentation time. I thought that this parameter was significant with first version of Nextera, and that in Flex/DNA prep it did not matter.
I'm afraid that I might be forced to look for end-repair method instead of nextera, which is so convenient. But the quality of input material is something that I have no control of.
I also considered the FFPE repair kits - is it worth a shot? There were publications proving that it greatly improves the results of microarray analyses. I'm not sure if it will do the job in case of NGS.
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The major challenge in working on FFPE extracted DNA is the low quality (and quantity) of the input DNA. Please make double sure that you are using sufficient amount of input DNA. If your genomic DNA is degraded, yet the average size is >500 bp on an agarose gel, then you can proceed with the same library prep kit you are using. *But you will have to reduce tagmentation time!!*
On the other hand, if your genomic DNA is highly degraded, my experience suggests not to use this kit, rather use the kit which follows end-rapair DNA --> adding of dA-tail ---> adaptors ligation workflow.
The major challenge in working on FFPE extracted DNA is the low quality (and quantity) of the input DNA. Please make double sure that you are using sufficient amount of input DNA. If your genomic DNA is degraded, yet the average size is >500 bp on an agarose gel, then you can proceed with the same library prep kit you are using. *But you will have to reduce tagmentation time!!*
On the other hand, if your genomic DNA is highly degraded, my experience suggests not to use this kit, rather use the kit which follows end-rapair DNA --> adding of dA-tail ---> adaptors ligation workflow.
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