Can I use sample DNA other than cfDNA with this kit?
While the cfDNA library preparation Kit has been validated to work with cfDNA from blood samples, the kit can also be used with low input DNA samples that do not need fragmentation. Additionally, the kit has been tested with input DNA derived from FFPE material. Below are details from an experiment involving FFPE samples.
DNA extraction was performed on FFPE curls from 4 sample sources (HG002, HG003, HG004, and Structural Multiplex Standard) using a Qiagen QIAmp FFPE DNA Extraction Kit. The DIN score for extracted DNA was between 3-5 (moderate). DNA was then fragmented with Covaris Ultrasonica Fragmentation targeting 175-350 bp.
Two replicates of each sample (n = 8) went through the cfDNA library preparation protocol with an input of 30 ng DNA. Ligation was performed using Twist Universal Adapters and the number of PCR cycles was set to 8. The library yield was 40-60 ng/μl.
Libraries underwent an 8-plex capture using the standard hybridization v2 protocol with the Twist ONCO DNA Profiler (~1.3 Mb) panel. The number of PCR cycles was set to 8, which resulted in a final yield of 2.14 ng/μl.
Sequencing results are shown in the table below.
| Sample | PCT_OFF_BAIT | HS_LIBRARY_SIZE | MEAN_TARGET_COVERAGE | ZERO_CVG_TARGETS_PCT | PCT_EXC_DUPE | FOLD_80_BASE_PENALTY | PCT_TARGET_BASES_30X | AT_DROPOUT | GC_DROPOUT |
|---|---|---|---|---|---|---|---|---|---|
| GM24143_mother_C12 | 29.47% | 14,209,598 | 52.0 | 0.34% | 3.89% | 1.62 | 84.06% | 10.99 | 0.03 |
| GM24143_mother_G12 | 29.65% | 13,576,332 | 52.2 | 0.32% | 4.04% | 1.63 | 83.72% | 11.51 | 0.03 |
| GM24149_father_B12 | 32.41% | 18,984,275 | 53.4 | 0.34% | 2.77% | 2.81 | 64.36% | 23.95 | 0.01 |
| GM24149_father_F12 | 32.72% | 19,094,135 | 54.4 | 0.36% | 2.72% | 2.86 | 64.14% | 24.61 | 0.01 |
| GM24385_son_A12 | 29.34% | 15,244,032 | 52.3 | 0.34% | 3.66% | 1.54 | 85.90% | 8.66 | 0.03 |
| GM24385_son_E12 | 29.39% | 15,378,322 | 53.4 | 0.32% | 3.62% | 1.52 | 86.96% | 8.78 | 0.03 |
| StrMultiRef_D12 | 28.65% | 7,124,970 | 48.8 | 0.34% | 7.53% | 1.44 | 88.81% | 1.71 | 0.95 |
| StrMultiRef_H12 | 28.81% | 7,592,887 | 48.7 | 0.36% | 7.02% | 1.43 | 88.68% | 1.72 | 0.97 |
| Average | 30.05% | 13,900,569 | 51.9 | 0.34% | 4.41% | 1.86 | 0.81 | 11.49 | 0.26 |
These results show that the cfDNA library preparation kit can be used on FFPE material and adapted for solid tumor workflows. Note that DNA fragmentation is still necessary even for samples with a DIN score <2. Additionally, DNA input ranges will need to be increased to accommodate the availability of DNA from FFPE extraction (~100-500 ng per 15 μM curl).
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