The QuantSeq-Pool protocol yields library fragments longer than 600 bp, with inserts longer than 430 bp when prepared from high quality Universal Human Reference RNA (UHRR). Therefore, QuantSeq-Pool libraries are compatible with all common sequencing read lengths.
Library shape and average insert size may vary, depending on the type of input sample (e.g., organism and/or cell type).
Libraries may also appear different depending on the microfluidics platforms used for quality control e.g., in TapeStation traces, the shape of the electropherogram curves appears shorter than in Bioanalyzer traces from the same libraries (see Fig. 1Figure, below).
Figure 1 | : Bionalyzer and TapeStation traces from the same libraries. Library 1: 8x 10 ng UHRR per Second Strand Synthesis (SSS) reaction, 14 cycles, Library 2: 88x 10 ng Human Neural Progenitor Cells (hNPC) RNA per SSS, 11 cycles. Both libraries were run in a Bioanalyzer High Sensitivity DNA Chip (left plots) and in a TapeStation High Sensitivity D5000 ScreenTape (left right graphs). Average Size was calculated from 170 – 3500 bp using the region analysis feature from the software specific to each microfluidics device.
The electropherograms from TapeStation and Bioanalyzer platforms are not equivalent due to the different properties of their gel matrixes and the units displayed on the Y axis (Fluorescent Units (FU) for Bioanalyzer and Normalized FU in TapeStation).
Therefore, we encourage customers it is important to not only to look at the shape of the electropherogram curves, but also at the Average Size of the libraries when assessing the quality of QuantSeq-Pool libraries. For more information, see QuantSeq-Pool Sample-Barcoded 3' mRNA-Seq User Guide, Typical Results, Appendix Einformation.
If you have are unsure about the size distribution of your libraries, please contact support@lexogen.com.