Next-generation sequencing (NGS) is being utilized for numerous new and exciting applications, such as single cell analysis, liquid biopsy research, metagenomics and targeted sequencing. Targeted sequencing has become a powerful approach by achieving high coverage of the region of interest while keeping the cost of sequencing and the complexity of data interpretation manageable.
Confident detection of single nucleotide variants (SNVs) present at 5% fraction, on average, typically requires a sequencing depth of >1000 reads (1). Therefore, high sequencing coverage is one of the critical factors for discovering cancer mutations present at low fractions, especially for detecting low-frequency variants.
Currently widely used methods for target enrichment before NGS runs include hybridization capture from sequencing libraries using target specific probes, as well as PCR amplification directly from sample DNA using target-specific primers (2). The PCR-based enrichment approach is well accepted, as the PCR process is easier to handle, requires less overall time, can easily accommodate much lower DNA input, and is more specific in terms of target sequence enrichment.
Despite these benefits, however, existing PCR-based target enrichment approaches introduce errors due to PCR amplification bias and artifacts. These errors significantly affect quantification accuracy and limit the ability to confidently detect low-frequency DNA variants.
To tackle the problems of PCR duplication and biased amplification in NGS analysis, a digital sequencing approach has been developed: the unique molecular indices (UMIs). With UMIs, each unique DNA molecule is barcoded before any amplification takes place. Sequence reads having different barcodes represent different original molecules; while sequence reads having the same barcode are results of PCR duplication from one original molecule.
Although molecular barcoding cannot prevent PCR duplication from happening, it does provide a convenient solution for tracking duplicates and treating them differently for downstream analysis. By employing UMIs, polymerase artifacts generated during PCR can be distinguished from sequence variants present in original molecules. With UMIs, not only does the accuracy of detection increase, but also the target quantification can be better achieved by counting the number of UMIs in the reads, rather than counting the number of total reads.
At QIAGEN, we have developed and optimized a high multiplex PCR amplicon sequencing process, which can accommodate thousands of target specific primers to enrich UMI-containing libraries in a single reaction (3). By utilizing UMIs, the newly released QIAseq Targeted DNA Panels offer a unique digital DNA sequencing approach, enabling confident detection of low-frequency DNA variants and increasing your chance of making the next groundbreaking discovery!
The workflow of QIAseq Targeted DNA Panels is shown in the following diagram:
Isolated DNA (as low as 20 ng) is enzymatically fragmented to generate small pieces of dsDNA. This step is followed by the library construction step, during which adapters with UMIs and sample indexes are incorporated into the unique DNA molecules generated in the previous step. Library fragments now serve as templates for target enrichment using single primer extension. In this step, targets are enriched using a single gene-specific primer and a universal forward primer.
The final step is library amplification and sample indexing (for Illumina® dual indexing only). After sequencing, UMIs enable the differentiation of true variant (red asterisk) from false positive (blue asterisk) for sensitive variant detection.
Would you like to learn more about this technology? Join us for a 2-part webinar series on September 1st and 8th.
1. September 1: Digital Next-Generation Sequencing for Targeted Enrichment, an Introduction to Technology – Watch the recording [Click here]
2. September 8: Digital DNAseq Technology – Targeted Enrichment for Cancer Research – Watch the recording [Click here]
In addition to the QIAseq Targeted DNA Panels, and using the same UMIs approach, QIAGEN has developed digital RNA-seq panels for gene expression analysis and RNAscan panels for sequencing fusion genes. Check out the digital NGS portfolio below.
QIAseq Targeted DNA Panels – Digital DNA sequencing to confidently detect low-frequency variants
QIAseq Targeted RNAscan Panels – Applying digital RNA sequencing to scan for known and novel fusion genes
QIAseq Targeted RNA Panels – Digital RNAseq for gene expression profiling
Want to talk to our NGS experts? Contact us at QIAseq.NGS@qiagen.com.
- 1. Spencer, D.H., et al. (2014) Performance of common analysis methods for detecting low-frequency single nucleotide variants in targeted next-generation sequence data. J Mol. Diagn. 16, 75–88. (Link)
2. Gnirke, A., et al. (2009) Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol. 27, 182–9. (Link)
3. Peng, Q., Satya, R.V., Lewis, M., Randad, P. and Wang, Y. (2015) Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes. BMC Genomics 16, 589. (Link)
Trademarks: Illumina® (Illumina, Inc.)