NGS Success - Post-Capture PCR
View next generation sequencing (NGS) tips for post-capture PCR:
- Use the appropriate indexes and the low multiplexing guidelines if using a lower (1-8) sample batch for sequencing. Due to the sequencing chemistry indexes must be sufficiently unique in order to maintain colour balance and facilitate accurate imaging by the sequencer.
- If QC yields are lower than expected check the tape station results. If libraries peak in the optimal area (250-350bp) but have a low yield, they can still be sequenced but may exhibit higher duplication rate and low loading patterns.
- If QC yields are higher than expected it is likely that the hybridisation and/or wash was not efficient and the captured libraries contain a high amount of off-target DNA. We recommend repeating the hybridisation and wash.
Figure 1. Analysis of amplified captured DNA using an Agilent High Sensitivity D1000 ScreenTape assay. The Blue electropherogram trace shows a peak in the desired size range of approximately 250–350 bp (+/– 10%). The green and yellow traces show examples of low DNA yield and failed PCR2.
- If high molecular weight fragments are observed in the TapeStation results, we recommend repeating the hybridisation and wash. The large fragments are indicative of sub-optimal enrichment and over-amplification during PCR.
Fig 2. Agilent High Sensitivity D1000 ScreenTape assay showing High Molecular weight fragments at ~500 bp.