PCR library amplification was performed prior to size selection in order to preserve the diversity of the library. Amplified library was eluted in 50 µL of elution buffer. Ten percent (5 µL) was set aside for QC and/or troubleshooting, and the remaining 45 µL were used for size selection.
The number of PCR cycles used to amplify each library is listed in Table 1.
To obtain size-selected library material in the 200–500 bp range, 0.6 volumes of SPRI beads were used to remove fragments larger than 500 bp. To the supernatant (containing fragments <500 bp), 0.2 volumes of beads (relative to the starting volume of 45 µL) were added, to exclude fragments <200 bp.
Sequencing-ready library was eluted in 30 µL of elution buffer, after which 1 µL of each library was used to assess quality and concentration on the Agilent 2100 Bioanalyzer.
Bioanalyzer data obtained from the analysis of 1 µL of each of the amplified, size-selected libraries are shown in Figure 3. Libraries were obtained from fragmented ChIP DNA or cDNA, with library construction input ranging between 1 and 200 ng. The maximum number of PCR cycles used during library amplification was 12 (for 1 ng ChIP DNA). The number of PCR cycles was progressively reduced to compensate for increasing amounts of input DNA (Table 1). These data demonstrated that the Eppendorf epMotion 5075 TMX is capable of fully automating the NGS library preparation process (from end repair to sequencing-ready library). Overlaid electropherograms showed that efficient size selection was achieved across the whole range of libraries, despite the variable amounts of amplified DNA in each sample.Table 2.
Yields of amplified, size-selected libraries are summarized in Table 3. These numbers represent net yields, after all unavoidable sample losses, attributable to one or more of the following possible factors:
DNA adsorption to plastic surfaces during pipetting and incubations.
Liquid handling (i.e. viscous solution carry-over)
Imperfect efficiency of enzymatic reactions
Incomplete elution of DNA from SPRI beads (five bead cleanup steps in total).
Imperfect PCR efficiency.