Expert view: Overcoming NGS obstacles

How can researchers get high-quality data from low-quality samples?

Targeted sequencing with unique molecular indices (UMIs) can be used to remove PCR bias and sequencing artifacts during library construction and sequencing. This is especially important with highly fragmented or limited amounts of usable sample, and extra rounds of PCR amplification are needed to generate a library.

What can be done to avoid variant drop out?

For DNA-based variant detection, ‘drop-outs’ can often be a problem with the design of the capture probes or amplicon sequencing primers in high – or low – GC regions of the genome. QIAseq Targeted DNA Panels use single primer extension (SPE) to enable high capture efficiencies in difficult-to-PCR regions. This, in combination with unique amplification chemistry and bioinformatics, allows QIAGEN to achieve full coverage on difficult-to-sequence genes.

What types of analysis tools are available?

QIAseq panels are supported through the GeneGlobe Data Analysis Centre and locally using CLC Biomedical Genomics Workbench. The online data analysis centre automatically performs the alignment, interprets UMIs, provides the sequencing summary and calls variants. In conjunction with UMIs, it can call variants down to 1% variant allele frequency. Biomedical Genomics Workbench is a comprehensive software package for the analysis and visualisation of data from all major next-generation sequencing platforms. It seamlessly integrates into a typical NGS workflow and is available for Windows, Mac OS X and Linux platforms. Incorporating cutting-edge technology and algorithms, Biomedical Genomics Workbench supports key next-generation sequencing features within genomics, transcriptomics and epigenomics research fields.