The xGen™ cfDNA & FFPE DNA Library Prep v2 MC Kit empowers you with highly complex variant identification from degraded and low-input research samples. The kit’s proprietary ligation strategy maximizes conversion and significantly reduces adapter-dimer formation. The unique molecular identifier (UMI) sequences incorporated during single-stranded ligation enable a variety of deduplication and error correction strategies used in research. It allows variant identification at ≤1% variant allele frequency (VAF).
The kit produces next generation sequencing (NGS) libraries suitable for many research applications that use degraded samples, including:
- Low-frequency, somatic variant identification of SNPs
- Insertions/deletions (indels)
- Identification of inherited germline SNPs and indels
- Whole genome sequencing
The protocol takes about 4 hours and includes only four major steps, minimizing sample handling:
- End repair. The End Repair Enzyme Mix converts cfDNA, or sheared, input DNA such as FFPE DNA into blunt-ended DNA that is ready for ligation.
- Ligation 1. The Ligation 1 Enzyme catalyzes the single-stranded addition of the Ligation 1 Adapter to only the 3′ end of the insert. This novel enzyme is unable to ligate inserts together, minimizing the formation of chimeras. The 3′ end of the Ligation 1 Adapter also contains a blocking group to prevent adapter-dimer formation.
- Ligation 2. The Ligation 2 Adapter acts as a primer to gap-fill the bases complementary to the Ligation 1 Adapter, followed by ligation to the 5′ end of the DNA insert to create a double-stranded product.
- PCR amplification. The xGen 2x HiFi PCR Mix is included to perform indexing PCR (not included) for Illumina® sequencing.
The kit includes all the reagents required for End Repair, Ligation 1 and Ligation 2 reactions, and PCR. Indexing primers (xGen UDI Primers) are supplied separately.
The kit was designed to work seamlessly with xGen hybridization capture probes and reagents.
