The SMARTer ThruPLEX Tag-seq Kit is a next-generation sequencing (NGS) library preparation kit designed to distinguish low-frequency variants in plasma, tissue, and formalin-fixed paraffin-embedded (FFPE) samples. The kit uses our proprietary SMARTer ThruPLEX chemistry to prepare molecularly tagged and sample-indexed Illumina NGS libraries from cDNA, cell-free DNA (cfDNA) and fragmented double-stranded DNA (dsDNA) (Figure 1).
SMARTer ThruPLEX Tag-seq features an extremely simple and fast workflow that can be completed in approximately 2 hours. It requires no intermediate purification or sample transfer steps (Figure 2), which are known to decrease library yield and increase the probability of contamination and handling errors.
When combined with hybridization-based target enrichment, SMARTer ThruPLEX Tag-seq libraries can be sequenced at great depth (5,000–10,000X raw coverage) to confidently detect variants present at low frequencies with only 1–50 ng of input DNA. SMARTer ThruPLEX Tag-seq provides more than 16 million random molecular tags to uniquely label the input DNA fragments (Figure 3). The unique molecular tags (UMTs) are incorporated during the ligation step, allowing subsequent computational correction of errors accumulated during amplification and sequencing. The construction of consensus sequences of molecular duplicates during bioinformatic analyses increases the accuracy of sequencing individual input molecules, enabling the detection of low-frequency variants with high sensitivity and specificity.
To validate the increased accuracy of variant detection using the SMARTer ThruPLEX Tag-seq Kit, we measured the limits of variant detection using reference cfDNA engineered with variants at different allele frequencies. This effort involved the steps of library preparation, target enrichment, sequencing, and data analysis (Figure 4). It demonstrates the utility of SMARTer ThruPLEX Tag-seq as a powerful tool for the detection of rare alleles.