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A variety of strategies are currently under investigation to develop further the display capabilities of AcMNPV and to improve the complexity of library that might be accommodated.
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A lower number of PCR cycles during library construction is desired to prevent the introduction of PCR biases and consequently reduced complexity of libraries.
As expected from the limited efficiency of the library construction protocol, the complexity of each library (defined as the numbers of unique molecules sequenced) was much lower than for the short-insert libraries.
Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones within the library, the more likely the library contains sequences that will bind a given target with useful affinity.
The complexity of a library is typically measured as the number of distinct molecules in the library (Daley and Smith 2013).
HeLa cells were infected with an MOI of 0.05, and the HeLa cell library was obtained following selection with 1 μg ml−1 puromycin; the complexity of the library was estimated to be about 2 × 10.
A comparison of the alternate tag extractions from the first bead library (1.74 M tags) suggests that sequencing length has an effect on the complexity of the library: the longer the tag sequence, the smaller the number of unique tags that are sequenced (Table 1).
Each sub-library contains independent and different random barcodes and helps preserve the high complexity of the library.
To verify if the complexity of the library was altered because of an insufficient initial quantity of input RNA, we prepared a library starting from 5 ng of total RNA.
For complex gene library cloning where sufficient numbers of clones need to be obtained in order to maintain the complexity of the library, more cycles of CPEC reaction might be needed.
For example, the use of a normalized AS RNA-Seq library preparation protocol or the introduction of a greater amount of input RNA may increase the complexity of the library, possibly enabling higher sensitivity as a result.
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