cfDNA low input (1 ng) hypomethylation enrichment DMN-seq, tumor, replicate 2
Patient samples and unidentified data were supplied under The University of Chicago protocol (IRB#10-209-A). The protocol involves data and sample collection from newly diagnosed patients with nonmetastatic colorectal cancer and noncancer controls undergoing colonoscopies that are negative at the University of Chicago Medical Center (UCMC). Blood from patients with CRC was collected prior to surgical operation via EDTA-containing vials and processed within six hours for plasma. Biopsies of primary tumors and adjacent normal-appearing colonic mucosa were also collected and stored in RNAlater solution (Thermo Fisher). Plasma and tissues in RNAlater were stored at -80 C before gDNA and cfDNA extraction. gDNA was extracted via an AllPrep DNA/RNA/miRNA Universal Kit (Cat. No. 80244) following the manufacturers protocol. In brief, the tissue was removed from the RNAlater solution for lysis and homogenization, and the DNA was collected by transferring the lysate through an AllPrep DNA Mini spin column (Qiagen). cfDNA was extracted from 1 mL of plasma via QIAamp Circulating Nucleic Acid Kit (Qiagen) following the instructions of the protocol.   A total of 10 ng of gDNA from CRC patient cohort tumor tissue and adjacent healthy colon tissue was used as the untreated sample, whereas 40 ng was used as the DME-treated sample. 1 replicate was used for CRC cohort gDNA. All gDNA samples were prepared, fragmented, and end-repaired in the same way as those used for methylated DNA enrichment. 1 ng, 0.5 ng. A total of 0.1 ng of cfDNA from CRC patients and healthy controls was used as the untreated sample, whereas 2 technical replicates of the same input level were used as DME-treated samples. The cfDNA samples were directly end-repaired without fragmentation. All DNA samples were then ligated to individual NEXTFLEX Unique Dual Index Barcodes (Revvity) following the recommended concentration on the basis of the input amount. After 0.8x Ampure bead purification, all the DME-treated samples were pooled on the basis of DNA type and concentration, i.e., 40 ng of CRC and healthy gDNA samples were pooled together, and cfDNA duplicates were pooled at each input level. All pooled samples were primer extended using R2 primer (IDT, Table S1) and Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and cleaned with 1.2  Ampure beads. DME treatment was conducted as described previously, followed by heat denaturation at 95 C for 5 min and 0.8x Ampure bead purification. All the DME-treated and untreated samples were then amplified in Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEXTFLEX Primer Mix 2.0 (Revvity). Finally, all the libraries were cleaned with 2 rounds of 0.8X AMPure XP beads, and their quality was examined via a high-sensitivity fragment analyzer at the University of Chicago Genomics Core Facility. All libraries were then sequenced with the Illumina NovaSeqX platform at the University of Chicago Genomics Core Facility.