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xGen™ cfDNA & FFPE DNA Library Preparation Kit

High library complexity from low quality samples

The xGen cfDNA & FFPE DNA Library Preparation Kit empowers you with accurate variant identification from degraded and low-input research samples.

xGen NGS—made for cfDNA & FFPE DNA library preparation.

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  • Get high conversion rates compared to TA-ligation-based methods with novel ligase and highly modified adapters
  • Identify variants at ≤1% variant allele frequency (VAF)
  • Get data from even highly degraded research samples

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.

Figure 1. Workflow for the xGen cfDNA & FFPE DNA Library Prep Kit. In an initial step, end repair enzymes convert cfDNA or sheared, input DNA into blunt-ended DNA ready for ligation. Then, a Ligation 1 Enzyme catalyzes the single-stranded addition of a Ligation 1 Adapter to the 3′ end of the insert. This novel enzyme is unable to ligate inserts together, which minimizes chimera formation. The 3′ end of the Ligation 1 Adapter also contains a blocking group to prevent adapter-dimer formation. The Ligation 2 Adapter acts as a primer to gap-fill bases complementary to the Ligation 1 Adapter, followed by ligation to the 5′ end of the DNA insert, to create a double-stranded product. In a final step, PCR incorporates sample index sequences 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.

Feature Details
Sample types High-quality DNA, cfDNA, DNA from FFPE samples, or double-stranded cDNA
Input range 1–250 ng
Adapters Included in the kit
Indexing primers (not included) xGen UDI Primers*
PCR amplification reagents (not included) KAPA Biosystems® HiFi HotStart ReadyMix, or equivalent
Compatible sequencing platforms Illumina® sequencing instruments
Compatible hybrid capture blockers xGen Universal Blockers TS

*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.

Figure 2. Overview of the hybridization capture and sequencing research workflow.

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).

Figure 3. In this example, the xGen cfDNA & FFPE DNA Library Prep Kit delivers higher conversion rates, complexity, and coverage. Libraries (n=12 for each DNA library prep kit) were generated according to manufacturer’s instructions with 10 ng of cfDNA reference standards, then captured with a 61 kilobase xGen Custom Hyb Panel. Libraries were pooled and sequenced on an Illumina® NextSeq® 500 instrument. Reads were mapped using the Burrows-Wheeler Aligner (BWA, 0.7.15). Coverage and complexity (estimated unique molecules; HS library size) were calculated using Picard (2.18.9). Relative conversion rates were calculated from mean target coverage at very high duplication rates.

Detect low frequency variants if cfDNA samples

Mutation EXPECTED VAF xGen cfDNA & FFPE DNA Library Prep Kit  Vendor A Vendor B
 EGFR:V769-D770insASV .25% 0.19% (3/3) 0.25% (3/3) 0.3% (2/3)
 EGR:E746-A750 0.11% (3/3) 0.3% (1/3) 0.26% (1/3)
 NRAS:A59T 0.22% (3/3) 0.74% (3/3) 0.37% (1/3)
 NRAS:Q61K 0.5% (3/3) 0.81% (3/3) 1.1% (3/3)
 EGFR:T790M 0.26% (2/3) 0.33% (3/3) 0% (0/3)
 EGFR:L858R 0.29% (3/3) 0% (0/3) 0.53% (0/3)

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.

Figure 4. In this example, the xGen cfDNA & FFPE DNA Library Prep Kit delivers higher library yield and complexity from FFPE samples across a range of sample qualities. Libraries (n=4 for xGen cfDNA & FFPE DNA Library Prep and Supplier A, n=3 for Supplier B) were generated according to the manufacturer’s instructions from 25 ng of Horizon FFPE reference standards with various qualities (HD798, HD799, HD803). Libraries were then captured with a 61 kilobase xGen Custom Hyb Panel and sequenced on an Illumina® NextSeq® 500 instrument. Reads were mapped using BWA (0.7.15). After sequencing, variants were called using VarDict (1.5.8) and the number of unique molecules (HS library size) was calculated using Picard (2.18.9).

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.

 Low quality FFPE
Variant Expected VAF 25 ng 100 ng 250 ng
EGFR:G719S 24.5 22.3 (3/3) 21.8 (3/3) 21.4 (3/3)
PIK3CA:H1047R 17.5 18.3 (3/3) 17.9 (3/3) 18.3 (3/3)
KRAS:G13D 15 13.5 (3/3) 13.2 (3/3) 13.2 (3/3)
NRAS:Q61K 12.5 11.8 (3/3) 10.2 (3/3) 10.3 (3/3)
BRAF:V600E 10.5 9.9 (3/3) 9 (3/3) 9.7 (3/3)
cKIT:D816V 10 9.8 (3/3) 8.6 (3/3) 9 (3/3)
PIK3CA:E545K 9 6.3 (3/3) 5.7 (3/3) 5.6 (3/3)
KRAS:G12D 6 5.9 (3/3) 5.1 (3/3) 5.3 (3/3)
EGFR:L858R 3 3.9 (3/3) 3.4 (3/3) 3.7 (3/3)
EGFR:E746-A750 2 0.6 (3/3) 0.2 (3/3) 0.4 (3/3)
EGFR:T790M 1 1 (3/3) 0.9 (3/3) 0.9 (3/3)

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.

Figure 5. In this example, xGen cfDNA & FFPE DNA Library Prep Kit delivers even GC coverage across the human genome. Whole genome libraries (n=44) were generated with NA12878 genomic DNA and sequenced on an Illumina® MiSeq® instrument. Reads were mapped using BWA (0.7.15), and normalized coverage was calculated using Picard (2.18.9).

*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

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