Minimal/Molecular residual disease (MRD) refers to the small number of tumor cells which may remain within a patient after therapeutic intervention. The detection of these remnants and monitoring of their abundance is a promising prognostic marker to identify individuals at risk of recurrence or in need of adjuvant therapy. Due to the low abundance of ctDNA present in samples obtained during remission, MRD assays need to be highly sensitive. In addition, each individual will have a different set of somatic variants, requiring personalized solutions for detection. Therefore, personalized NGS assays with high sensitivity and specificity are needed for MRD diagnostics. To address this need and empower accurate assessments of MRD, Twist Bioscience has developed the MRD Rapid 500 Panels. This product enables customers to design, manufacture and ship fully personalized MRD panels (up to 500 targets) in as little as six days. To demonstrate the detection sensitivity of Twist Rapid 500 MRD panels, we designed five custom MRD panels which specifically target somatic variants found in Breast, Lung, CRC, Melanoma and Renal Cell Carcinoma. Each of these MRD panels were designed to include 197 targets, with 3-5 variants per tissue origin and a selection of passenger mutations. Probe sequences of each panel were designed to incorporate the variant allele in the test sample set. To create the sample set, we blended synthetic variant sequences with fragmented cell line gDNA (NA12878) to form a contrived specimen which approximates the profile cell-free and circulating tumor DNA. Five frequency levels were created with average variant allele frequencies (VAFs) of 0% (WT), 0.01%, 0.05%, 0.1% and 2%. Libraries were prepared with UMI adapters and target enrichment was performed using the MRD panels.    With a sequencing depth of 80,000x, variant calling results revealed that an average of 20 SNV targets can be detected with confidence in the 0.01% VAF samples for each MRD panel, clearly distinguishable from the WT control samples. In addition to demonstrating the accuracy of variant calling by targeting the alternate allele, we showcase the utility of targeting a large number of variants for the detection of an MRD signature at very low levels (e.g. 0.01% VAF).   In summary, the performance of the Twist MRD Rapid 500 Panels showed high detection sensitivity of ultra low-frequency somatic mutations.

Introduction DNA methylation at CpG nucleotide sites in eukaryotes is a key epigenetic mark that helps regulate gene expression. Specific changes in CpG methylation occur in many human cancers, making them a promising biomarker for early cancer detection. However, existing assays can be costly, lack specificity to regions of interest and often provide only semi-quantitative estimates of the methylation fraction. Here, we present new tools to address these challenges including the use of a targeted methylome panel to decrease costs associated with next generation sequencing (NGS), methylation controls to calibrate quantitative assays and UMIs for accurate deduplication in low-diversity samples. Experimental Procedures Genomic DNA (gDNA) was prepared for sequencing using the Twist Methylation Detection System consisting of enzymatic methyl conversion library generation and hybrid capture using the Twist Human Methylome Panel. Twist CpG methylation specific controls were spiked into gDNA with different methylation rates prior to library preparation and taken through the Twist Methylation Detection System using homologous panels to demonstrate how these controls can be used to calibrate detection assays. Additionally, libraries were generated using cell free DNA (cfDNA) and either conventional or UMI-containing adapters during the library preparation ligation step to investigate the impact on quantitative detection and total unique coverage. Data Using the Twist Human Methylome Panel at 150x raw coverage achieves uniform coverage with low off-bait Picard metrics. 6.59 million CpG sites were detected using a minimum depth of 10X. Target capture with the Human Methylome Panel allows for informative CpG calling of up to 82 samples on a single Illumina Novaseq S4 flowcell. In contrast, performing traditional whole genome bisulfite sequencing (WGBS) without target enrichment, only 3 samples could be run on the same flowcell. The Twist CpG methylation specific controls are constructed of 48 unique contrived sequences that contain a total of 8 different levels of methylation, ranging from 100% to 0%. Including these controls allows for quantitation of methylation levels in the experimental samples qualification of the enzymatic conversion process. Conclusions Our study uses several new products found in the Twist methylation detection portfolio that interrogate genome-wide methylation patterns for various applications. Not only have we found ways to better control assays, but we have also determined methods to account for lower costs compared to WGBS. 

We probed genetic differences produced by a long-term selective breeding program by creating an F2 cross between two phenotypically divergent outbred rat lines and then used this F2 cross to map loci for the selection trait, sensation-seeking behavior, as well as several putatively correlated traits.