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.
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!