ecSeq Bioinformatics GmbH, Germany
Next-generation sequencing increasingly replaces traditional Sanger sequencing for routine genetic testing applications. The higher throughput allows higher sensitivities for detecting low-frequency DNA mutations. However, more sequence reads do not automatically lead to a higher sensitivity and accuracy. Technical limits grounded in NGS technology and the library preparation, such as PCR duplicates, low complexity and false positives, need to be addressed. This leads to an increase in the diversity and complexity of available commercial NGS sample preparation kits.
Recent kits targeting low-frequency variant detection (for oncology) combine approaches such a unique molecular identifiers, single primer extension and tiled amplicon designs to address these issues. We show how these approaches work, and what are the practical consequences of these approaches on the observed sequence reads and on the downstream bioinformatics analysis.
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