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xGen Prism DNA Library Prep Kit

High library complexity from low quality samples

The xGen Prism DNA Library Prep Kit empowers you with sensitive and accurate variant detection from degraded samples, such as cell-free DNA (cfDNA) or formalin-fixed, paraffin-embedded (FFPE) samples. The kit’s proprietary ligation strategy maximizes conversion and virtually eliminates adapter-dimer formation. The unique molecular identifier (UMI) sequences incorporated during single-stranded ligation enable a variety of deduplication and error correction strategies.

  • Get higher conversion rates compared to TA-ligation-based methods with novel ligase and highly modified adapters
  • Detect variants at ≤1% variant allele frequency (VAF)
  • Get data from even the most degraded samples
  • Minimize sequence errors with UMIs

Applications

The xGen Prism DNA Library Prep Kit produces high-quality next generation sequencing (NGS) libraries that are suitable for many applications including:

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

Method

The entire workflow takes about 3 hours. There are 4 major steps in this protocol (Figure 1):

  • End repair. The End Repair Enzyme Mix converts cfDNA or sheared, input 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 UMI, 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. Overview of the xGen Prism DNA Library Prep Kit process.

Technical details

The xGen Prism DNA Library Prep Kit includes all the reagents required for End Repair, and Ligation 1 and Ligation 2 reactions. Table 1 provides additional specifications for this kit.

Table 1. Specifications, additional reagents, and equipment

Feature Details
Sample types High-quality DNA, cfDNA, DNA from FFPE samples, or double-stranded cDNA
Fragmentation Mechanical shearing recommended, except for highly fragmented samples like cfDNA
Input range 1–250 ng
Adapters Included in the kit
UMIs 32 optimized, fixed, 8 bp UMIs
Indexing primers (not included) xGen UDI Primer Pairs*
PCR amplification reagents (not included) KAPA Biosystems® HiFi HotStart ReadyMix
Compatible sequencing platforms Illumina® sequencing instruments
Compatible hybrid capture blockers xGen Universal Blockers—TS Mix

*Contact applicationsupport@idtdna.com for help ordering other indexing designs or configurations.

Complete workflow for hybridization capture experiments

The xGen Prism DNA Library Prep Kit was optimized 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 you need.

Figure 2. Overview of hybridization capture sequencing workflow.

Superior conversion and UMI-based error correction enables ultra-low variant detection with cfDNA samples

The unique, single strand, ligation strategy and workflow of xGen Prism DNA Library Prep Kit delivers higher 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 cfDNA. A higher conversion rate translates to higher complexity and coverage than other DNA library prep kits using cfDNA (Figure 3). In addition, the xGen Prism DNA Library Prep Kit includes UMI-containing adapters that enable bioinformatic error correction. Combining higher complexity and coverage with a stringent error correction allows the xGen Prism DNA Library Prep Kit to deliver higher sensitivity and positive predictive value (PPV) when detecting ultra-low frequency variants (Figure 4).

Figure 3. The xGen Prism DNA Library Prep Kit delivers higher conversion rates, complexity and coverage. Libraries were generated according to the manufacturer’s instructions with 10 ng of cfDNA reference standards, then captured with a custom 61 kb xGen Lockdown Panel. Libraries were pooled and sequenced on an Illumina NextSeq® 500 instrument. Reads were mapped using 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.
Figure 4. The xGen Prism DNA Library Prep Kit enables higher sensitivity and specificity for ultra-low variant detection. Libraries 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 custom, 61 kb xGen Lockdown Probe Panel. After sequencing, reads were mapped using BWA (0.7.15). Start-stop deduplication or cc (collapsed combined) read families error correction was performed as described in the xGen Prism Analysis Guide. Finally, variants were called using VarDict (1.5.8); only variants with PASS/f0.02 filters were used for start-stop deduplicated data, while no filters were applied for cc read families error corrected xGen Prism data. PPV = positive predictive value.

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

Analysis of FFPE samples has its own unique challenges, including difficulty successfully generating libraries from samples of variable quality, especially when inputs are limiting. The xGen Prism DNA Library Prep Kit leverages its higher conversion rate to give higher library yields from low inputs of even severely damaged FFPE samples (Figure 5). Higher conversion rates and yields translate to higher library complexity (Figure 5), which can increase confidence in variant calling. With severely damaged FFPE samples, the xGen Prism DNA Library Prep Kit reliably delivers sensitive SNP and indel detection across a range of inputs. We generated libraries using a severely damaged FFPE reference standard with variants of defined allele frequencies, then captured with a 238 kb xGen Lockdown 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 detected. Each variant was detected in all replicates for the sample input at a frequency comparable to the expected VAF (Table 2).

Figure 5. The xGen Prism DNA Library Prep Kit delivers higher library yield and complexity from FFPE samples across a range of sample qualities. Libraries were generated according to the manufacturer’s instructions from 25 ng of FFPE reference standards with various qualities. Libraries were then captured with a custom 61 kb xGen Lockdown Panel and sequenced on an Illumina NextSeq 500 instrument. Reads were mapped using BWA (0.7.15), and the number of unique molecules (HS library size) were calculated using Picard (2.18.9).

Table 2. xGen Prism DNA Library Prep Kit enables higher sensitivity and specificity of ultra-low variant detection.

 Severe 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

Whole genome sequencing data demonstrates that the xGen Prism DNA Library Prep Kit has even coverage across the human genome with little evidence of bias (Figure 6). Even coverage is made possible by the reduced number of PCR cycles required, due to higher conversion rates of input molecules.

Figure 6. xGen Prism DNA Library Prep Kit delivers even GC coverage across the human genome. Whole genome libraries 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).

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