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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 highly complex variant identification from degraded and low-input research samples.

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

Automation out of the box—no expertise necessary.

Automation of IDT's xGen™ cfDNA & FFPE DNA Library Prep Kit just got easier with the Biomek NGeniuS instrument. Request a consultation with one of our NGS experts to learn more.

xGen NGS—made for automation.

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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.

For customers using legacy xGen cfDNA & FFPE DNA Library Prep MC Kits, these products can be ordered here.

Christopher Noune
Australian Genome Research Facility Ltd

We've found the xGen 2x HiFi PCR Mix to be a high performing enzyme, demonstrated superior GC-bias and yielding nearly 2x more library than its competitors using the same number of cycles. This means that we can reduce the number of PCR cycles needed to achieve high yields from low inputs and reduce PCR duplicates, leading to an overall improvement in our IDT workflows.

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.

Figure 1. Workflow for the xGen cfDNA & FFPE DNA Library Prep v2 MC 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 which creates a double-stranded product. In a final step, PCR with the IDT 2x HiFi PCR mix incorporates sample index sequences for sequencing on Illumina® platforms.

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.

Feature Details
Sample types High-quality DNA, cfDNA, DNA from FFPE samples
Input range 1–250 ng
Adapters Included
Indexing primers (not included) xGen UDI Primers*
PCR amplification reagentsxGen 2x HiFi PCR Mix, included
Compatible sequencing platforms Illumina® sequencing instruments
Compatible hybrid capture blockers xGen Universal Blockers—TS Mix

*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.

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

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).

Figure 3. xGen cfDNA &FFPE DNA Library Prep v2 MC Kit delivers higher yields, complexity, and coverage. Libraries were prepared in triplicate according to the manufacturer’s instructions with 50 ng of Horizon cfDNA reference standard and 7 cycles of PCR. Following quantification, libraries were captured with a custom 61 kb (target space) xGen Hyb Panel using the xGen Hybridization and Wash Kit. 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, coverage and complexity were calculated.

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

MutationExpected VAFxGen cfDNA & FFPE DNA Library Prep v2 MC KitLibrary Kit ALibrary Kit B
EGFR: L858R0.25%0.13 (3/3)0.21 (3/3)0.21 (3/3)
EGFR: E746-A7500.25%0.11 (3/3)0.19 (3/3)0.12 (3/3)
EGFR: T790M0.25%0.29 (3/3)0.36 (3/3)0.12 (3/3)
KRAS: G12D0.32%0.33 (3/3)0.36 (3/3)0.33 (3/3)
NRAS: Q61K0.32%0.23 (3/3)0.31 (2/3)0.22 (3/3)
NRAS: A59T0.32%0.17 (3/3)0.43 (2/3)0.22 (3/3)
PIK3CA:E545K0.32%0.16 (3/3)0.11 (3/3)0.36 (3/3)

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).

Figure 4. Library yield and complexity from varying qualities of Formalin Compromised DNA (fcDNA) reference standards. Libraries (n = 4 each) were prepared in triplicate by three commercial library prep kits following each of the manufacturer’s protocols with 25ng total input. Following 10 cycles of PCR, libraries were quantified using a Qubit™ dsDNA HS Kit. Then, 500 ng of each library were captured using a custom 180 kb (target space) xGen Hyb Panel with the xGen Hybridization and Wash v2 Reagents and Beads, following the xGen Hybridization and Wash Kit v2 protocol with an overnight hybridization and 13 cycles of PCR. The captured libraries were then pooled equimolar and sequenced on a NextSeq 500 (Illumina), using a high output 300 cycle kit and the manufacturer's protocol. Samples were subsampled to 8 million paired end reads and the number of unique molecules (HS library size) was determined with Picard.

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

VariantExpected VAF 25ng50ng100ng250ng
EGFR: G719S24.523.9 (3/3)22.9 (3/3)22.1 (3/3)23.0 (3/3)
PIK3CA:H1047R17.516.9 (3/3)18.6 (3/3)17.5 (3/3)18.0 (3/3)
KRAS: G13D15.012.1 (3/3)13.2 (3/3)12.1 (3/3)12.0 (3/3)
NRAS: Q61K12.57.9 (3/3)11.3 (3/3)8.7 (3/3)8.9 (3/3)
BRAF: V600E10.511.2 (3/3)11.1 (3/3)11.0 (3/3)10.6 (3/3)
KIT: D816V10.08.0 (3/3)8.4 (3/3)7.7 (3/3)7.7 (3/3)
PIK3CA: E545K9.06.0 (3/3)7.0 (3/3)6.1 (3/3)6.3 (3/3)
KRAS: G12D6.05.7 (3/3)5.2 (3/3)6.3 (3/3)5.8 (3/3)
EGFR: L858R3.03.2 (3/3)2.9 (3/3)2.8 (3/3)2.5 (3/3)
EGFR: E746-A7502.00.8 (3/3)0.5 (3/3)0.8 (3/3)0.5 (3/3)
EGFR: T790M1.00.9 (3/3)0.7 (3/3)0.8 (3/3)0.9 (3/3)

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.

Figure 5. xGen cfDNA & FFPE DNA Library Prep Kit normalized GC coverage across the human genome from cfDNA. 10ng cfDNA libraries were generated using xGen cfDNA & FFPE DNA Library Prep v2 MC Kit, using cfDNA extracted from two healthy donors from BioChain. Libraries were generated in triplicate using the manufacturer’s protocol, with 11 cycles of PCR. Libraries were quantified with Qubit dsDNA HS Kit and average size was determined with the Tapestation 4200 High Sensitivity D1000 (Agilent). Libraries were then pooled equimolar and sequenced on a Nextseq 500 (Illumina), using a high-output 300 cycle kit following the manufacturer’s protocol. The cfDNA libraries were subsampled to 20 million paired end reads for analysis with <0.2% of the genome bin at <15% GC and <0.1% of the genome bin at >80% GC.

Frequently asked questions

*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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