ThruPLEX Tag-seq combines molecular tags with ThruPLEX chemistry to construct molecularly tagged and sample-indexed Illumina NGS libraries. Each kit contains more than 16 million unique sequences used to tag individual DNA fragments prior to amplification, providing tracking of the fragments through the library preparation, target enrichment, and data analysis processes to detect low-frequency alleles or count individual fragments. The ThruPLEX chemistry is engineered and optimized to produce highly diverse libraries with reproducible sequencing performance from 1 to 50 ng of DNA. The entire three-step workflow takes place in a single tube or well in about 2 hours. No intermediate purification steps and no sample transfers are necessary, which prevents handling errors and loss of valuable samples. The ThruPLEX Tag-seq Kit includes all necessary reagents including indexes for multiplexing up to 96samples. Once purified and quantified, the resulting library is ready for Illumina NGS instruments using standard Illumina sequencing reagents and protocols.
To benefit from the information provided by the unique molecular tags (UMT) in ThruPLEX Tag-seq libraries, the sequencing data must be processed to group reads into amplification families and construct consensus reads. Two bioinformatics solutions designed specifically for processing data from ThruPLEX Tag-seq libraries are available:
Connor is an open-source bioinformatics tool hosted on GitHub (https://github.com/umich-brcf-bioinf/Connor) for processing sequencing data generated from ThruPLEX Tag-seq libraries. Connor takes an aligned BAM file as input, processes the UMT information and generates a processed BAM file containing consensus sequences.The output BAM file can be used with variant callers, such as FreeBayes,VarScan2, or GATK HaplotypeCaller.
Curio is a cloud-based bioinformatics platform available through Curio Genomics. Sequencing data from ThruPLEX Tag-seq libraries can be easily uploaded, processed, and visualized. The platform features an ultra-fast and user-friendly interface and a powerful alignment viewer. The platform is also equipped with modules for performing variant detection, analysis, and visualization. For more information, visit www.curiogenomics.com.