xGen™ cfDNA & FFPE DNA Library Preparation Kit

Product details

Applications

The xGen cfDNA & FFPE DNA Library Prep MC Kit v2 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 (WGS)

The xGen cfDNA & FFPE DNA Library Prep protocol takes about 4 hours and includes only four major steps, minimizing sample handling (Figure 1):

• 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 (primers sold separately) for Illumina® sequencing.

Technical Details

The xGen cfDNA & FFPE DNA Library Prep v2 MC Kit includes all the reagents required for End Repair, Ligation 1 and Ligation 2 reactions, and PCR.

Table 1. Specifications, additional reagents, and equipment.

*Contact us for immediate assistance in ordering other indexing designs or configurations.

Complete workflow for hybridization capture research experiments

The xGen cfDNA & FFPE DNA Library Prep v2 MC Kit was designed to work seamlessly with xGen hybridization capture probes and reagents (Figure 2). Whether your project requires whole exome sequencing or custom panels, IDT has the capture solutions for you.

Product data

Comprehensive conversion and error correction enables ultra-low variant identification with cell-free DNA

The unique, single-stranded ligation strategy of the IDT xGen cfDNA & FFPE DNA Library Prep v2 MC Kit and workflow delivers high conversion of input DNA molecules to sequencing data. This high conversion rate is critical for identification of ultra-low frequency variants, which is common in the analysis of cell-free DNA (cfDNA). A higher conversion rate translates to more complexity and coverage than other DNA library prep kits for cfDNA (Figure 3). In addition, the xGen cfDNA & FFPE DNA Library Prep Kit includes adapters that contain unique molecular identifiers (UMIs), which enable bioinformatic error correction. Combining higher complexity and coverage with stringent error correction better enables the identification of ultra-low frequency variants (Table 2).

Table 2. xGen cfDNA & FFPE DNA Library Prep Kit identifies low frequency variants in NGS reference samples

Table 2. Libraries were prepared in triplicate from 50 ng input Horizon cfDNA reference standards using the xGen cfDNA &FFPE DNA Library Prep v2 MC Kit in addition to two other commercially available library prep kits. Libraries were then captured with a custom 180 kb (target space) xGen Hyb Panel targeting 7 identified SNPs using the using the xGen Hybridization and Wash v2 Reagents and Beads. Captured libraries were pooled and sequenced on a NextSeq 500 (Illumina), using a high output 300 cycle kit and the manufacturer's protocol. After subsampling to 85M total reads, the average variant allele frequency for each of the targeted mutations was calculated for each library prep kits using VarDict.

Higher coverage and complexity deliver reliable variant and indel identification in FFPE samples

Research analysis of FFPE samples has its own unique challenges, including difficulties in generating libraries from samples of variable quality or with low inputs. The xGen cfDNA & FFPE DNA Library Prep v2 MC Kit leverages high conversion rates to achieve high library yields from low inputs of even severely damaged FFPE research samples (Figure 4). Higher conversion rates and yields translate to higher library complexity (Figure 4), which can increase confidence in variant calling. With severely damaged FFPE samples, the xGen cfDNA & FFPE DNA Library Prep v2 Kit delivers SNP and indel identification across a range of inputs (Table 3).

Table 3. Observed Average Mutation Allelic Frequency of Severely Degraded FFPE DNA Library Input

Table 3. xGen cfDNA & FFPE DNA Library Prep v2 MC Kit enabled the mutation detection in severely degraded FFPE reference sample at varying library input. Each sample was captured with a custom 180 kb (target space) xGen Hyb panel targeting the verified mutations, using xGen Hybridization and Wash v2 Reagents and Beads, following the xGen Hybridization and Wash Kit v2 Protocol and sequenced to an average depth of 30 million read pairs on a NextSeq 500 (Illumina) using a high-output 300 cycle kit and the manufacturer's protocol. The average variant allele frequency (VAF) was calculated for each of the 11 mutations across all three replicates using VarDict, with a minimum variant allele depth of 3 single-stranded consensus reads. The number of replicates with a response is shown in parentheses.

Uniform GC coverage across the human genome

Internal whole genome sequencing (WGS) experiments demonstrated that the xGen cfDNA & FFPE DNA Library Prep v2 MC Kit has even coverage across the human genome with little evidence of bias (Figure 5), resulting in no significant GC bias for 99.7% of the human genome. Even coverage is made possible by the reduced number of PCR cycles required, due to higher conversion rates of input molecules.

Frequently asked questions