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. 

Fig. 1. Analysis of amplified captured DNA

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. High molecular weight fragments in TapeStationFig 2. Agilent High Sensitivity D1000 ScreenTape assay showing High Molecular weight fragments at ~500 bp.

 

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