xGen™ cfDNA & FFPE DNA Library Preparation Kit

Product details

Applications

The xGen cfDNA & FFPE DNA Library Prep Kit produces next generation sequencing (NGS) libraries suitable for many research applications, including:

• Low-frequency, somatic variant identification of single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) from cfDNA and FFPE samples
• Identification of inherited germline SNPs and indels from degraded samples
• Whole genome sequencing (WGS) from degraded samples

xGen cfDNA & FFPE DNA Library Prep protocol takes about 3 hours and includes four major steps, thereby 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 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, which minimizes 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. PCR is used to incorporate sample index sequences needed for sequencing on Illumina^® platforms.

Technical Details

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

Table 1. Specifications, additional reagents, and equipment.

*Contact us for help ordering other indexing designs or configurations.

Complete workflow for hybridization capture research experiments

The xGen cfDNA & FFPE DNA Library Prep 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 that you need.

Product data

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

The IDT xGen cfDNA & FFPE DNA Library Prep Kit’s unique, single-stranded ligation strategy and workflow delivers a high conversion of input DNA molecules to sequencing data. A high conversion rate is critical for the 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 enables the detection of ultra-low frequency variants (Table 2).

Detect low frequency variants if cfDNA samples

Table 2. In this example, the xGen cfDNA & FFPE DNA Library Prep Kit enables identification of ultra-low variants. Libraries (n=3 each) were generated according to the manufacturer’s instructions using 25 ng of a cfDNA reference standard with an allele frequency of 0.25%. Libraries were captured with a 61 kilobase xGen Custom Hyb Panel. After sequencing, reads were downsampled to 20M each and mapped using BWA (0.7.15).

Higher coverage and complexity deliver reliable variant and indel detection 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 Kit leverages high conversion rates to achieve high library yields from low inputs of even severely damaged FFPE research samples (Figure 5). 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 Kit delivers SNP and indel identification across a range of inputs.

Libraries were generated using a severely damaged Horizon FFPE reference standard with variants of defined allele frequencies, then captured with a 238 kb xGen Custom Hyb Probe Panel. After sequencing, reads were mapped using BWA (0.7.15), and variants were called using VarDict (1.5.8). The average observed variant allele detection reported by VarDict is shown along with the number of samples in which each variant was identified. Each variant was identified in all replicates for the sample input at a frequency comparable to the expected VAF (Table 3).

Table 3. xGen cfDNA & FFPE DNA Library Prep Kit enables sensitive detection of low frequency variants.

Uniform GC coverage across the human genome

Internal whole genome sequencing experiments demonstrated that the xGen cfDNA & FFPE DNA Library Prep Kit has even coverage across the human genome with little evidence of bias (Figure 5). Even coverage is made possible by the reduced number of PCR cycles required, due to higher conversion rates of input molecules.