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.
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.
Request consultationThe xGen cfDNA & FFPE DNA Library Prep Kit produces next generation sequencing (NGS) libraries suitable for many research applications, including:
xGen cfDNA & FFPE DNA Library Prep protocol takes about 3 hours and includes four major steps, thereby minimizing sample handling (Figure 1):
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.
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.
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.
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).
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) |
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.