Next Generation Sequencing Troubleshooting

Next Generation Sequencing (NGS) experiments are complex and comprise of multiple steps. Unlike Sanger sequencing, NGS DNA sequencing steps include sample enrichment, library preparation and complex computational studies. While sequencing kits help your experiments to run smoothly, with these many variables there is always the possibility that something goes wrong.

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Coverage Dropout

  • Poor DNA/RNA quality: The sample is degraded because of poor storage or was isolated from a Formalin-Fixed paraffin-embedded tissue sample

  • Small sized amplicons: Amplicons in your libraries should generally be around 300 bp

  • High GC content: Use a specific kit for High-GC content regions

Ambiguous Clustering Calls

  • Low diversity libraries / wrong quantification: The quantification may not have been executed precisely enough before building the library. Please check the quality of your quantification

  • Insufficient purity: Be sure to purify your DNA preparation before using it for library creation

When repeating your NGS experiments, be sure to include these tips to minimize the chance for mistakes

  • Use short DNA fragments

  • Purify your DNA to remove small fragments

  • Use only highly pure DNA


Hopefully with these tips your problems will quickly become a thing of the past. If you're still having issues - or have extra tips - let us know via the livechat function!