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.
The xGen Prism DNA Library Prep Kit produces high-quality next generation sequencing (NGS) libraries that are suitable for many applications including:
The entire workflow takes about 3 hours. There are 4 major steps in this protocol (Figure 1):
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).
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).
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)|
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.