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Hi All,
I've seen many posters warning about over amplification of ChIP-Seq libraries. But I wanted to get some clarification on want people mean by "over amplification". If you stop at 12-14 cycles and see good amplification, then the library is likely to have a high complexity. Obviously, if it takes more cycles to get good amplification, then you presumably had fewer good adapter-fragment ligation products and will end up with a less complex library. This is why it is worth monitoring amplification efficiency closely rather than just blindly doing 20 cycles on all libraries. I assume people mean that an "over-amplified" library is one where you had to perform so many cycles to get amplified product, that the complexity is likely to be low - is this how most people on this forum are using this term?
The alternative definition that I have seen for an "over-amplified" library, is where more cycles are used than are actually needed. For example, if a library was sufficiently amplified after 14 cycles, but an additional 6 cycles are performed, then in the final cycles you may be denaturing the amplified fragments and no productive amplification is actually occurring during those last cycles. In this later case, I would not anticipate that there would be a large impact on the complexity of the library. We have noticed that if we perform more amplifications than are needed, libraries sometime appear to have a larger size range when measured on a bioanalyzer (presumably due to daisy-chaining of adapter sequences, since those sequences can readily reanneal from a denatured complex library). However, the complexity of these libraries is fine when we sequence them (typically >95% of aligned reads are non-redundant at a sequencing depth of 40 million reads). Are there dangers/problems with performing more cycles than are actually needed for library amplification that I am missing?
I've seen many posters warning about over amplification of ChIP-Seq libraries. But I wanted to get some clarification on want people mean by "over amplification". If you stop at 12-14 cycles and see good amplification, then the library is likely to have a high complexity. Obviously, if it takes more cycles to get good amplification, then you presumably had fewer good adapter-fragment ligation products and will end up with a less complex library. This is why it is worth monitoring amplification efficiency closely rather than just blindly doing 20 cycles on all libraries. I assume people mean that an "over-amplified" library is one where you had to perform so many cycles to get amplified product, that the complexity is likely to be low - is this how most people on this forum are using this term?
The alternative definition that I have seen for an "over-amplified" library, is where more cycles are used than are actually needed. For example, if a library was sufficiently amplified after 14 cycles, but an additional 6 cycles are performed, then in the final cycles you may be denaturing the amplified fragments and no productive amplification is actually occurring during those last cycles. In this later case, I would not anticipate that there would be a large impact on the complexity of the library. We have noticed that if we perform more amplifications than are needed, libraries sometime appear to have a larger size range when measured on a bioanalyzer (presumably due to daisy-chaining of adapter sequences, since those sequences can readily reanneal from a denatured complex library). However, the complexity of these libraries is fine when we sequence them (typically >95% of aligned reads are non-redundant at a sequencing depth of 40 million reads). Are there dangers/problems with performing more cycles than are actually needed for library amplification that I am missing?