Assay Validation of Cell-Free DNA Shallow Whole-Genome Sequencing to Determine Tumor Fraction in Advanced Cancers.
Author(s): Rickles-Young M, Tinoco G, Tsuji J, Pollock S, Haynam M, Lefebvre H, Glover K, Owen DH, Collier KA, Ha G, Adalsteinsson VA, Cibulskis C, Lennon NJ, Stover DG
Publication: J Mol Diagn, 2024, Vol. , Page
PubMed ID: 38490303 PubMed Review Paper? No
Purpose of Paper
This paper describes the validation of an ultra low-pass whole genome sequencing (ULP-WGS) assay for identification of tumor fraction in cell-free DNA (cfDNA) and establishes the minimum tumor fraction by comparing results with whole exome sequencing (WES), identifies the minimum cfDNA input required, and investigates assay reproducibility. ULP-WGS metrics and tumor fraction were compared between matched blood specimens collected in Streck and EDTA tubes that were stored for up to 24 h at room temperature before analysis.
Conclusion of Paper
Using the ultra low-pass whole genome sequencing (ULP-WGS) assay, the mean normalized standard deviation (MNSD) of the tumor fraction increased as the tumor fraction fell below 3% but was low among specimens with a tumor fraction above 3% (≤0.54). When the tumor fraction was 3 or 4%, the sensitivity of mutations detected by whole exome sequencing was 97.2-100%. GC-corrected mean absolute deviation (MAD) scores were very similar between the two sequencing instruments and the data did not cluster by instrument during hierarchical clustering. Although the observed tumor fraction was consistent when the input to the ULP-WGS assay was as low as 1 ng DNA, some quality metrics were not met; consequently, the authors suggest the use of at least 20 ng input. Overall, quality metrics (mean depth, library size, or MAD) did not differ between matched specimens that were collected in EDTA and Streck tubes and stored for up to 24 h at room temperature. Tumor fractions were also generally comparable between EDTA and Streck specimens. For the three specimens stored <4 h at room temperature, there was a 4.75% absolute error between the tube types, but the absolute error was 7.9% for the 12 specimens stored 12-24 h. Based on these data, the authors suggest both tubes are acceptable for ULP-WGS and WES and that EDTA tubes can be stored for ≤8 h.
Studies
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Study Purpose
This study describes the validation of an ultra low-pass whole genome sequencing (ULP-WGS) assay for identification of the tumor fraction of cfDNA and establishes the minimum acceptable tumor fraction by comparing results with whole exome sequencing, identifies minimum cfDNA input and investigates assay reproducibility. ULP-WGS metrics and tumor fraction are compared between blood collected from the same patients into Streck and EDTA tube that were stored for up to 24 h at room temperature before analysis. Unless otherwise specified, blood was collected from thirty-four patients with metastatic breast cancer into EDTA tubes and plasma was separated within 4 h of blood collection by density gradient centrifugation (details not provided). Plasma was then recentrifuged at 19000 g for 10 min and frozen at -80°C until extraction. Plasma was thawed at room temperature, and cfDNA was isolated using the Circulating DNA Kit on the QIAsymphony liquid handling instrument. Ultra low-pass (ULP) shallow whole genome sequencing (WGS) libraries were sequenced using HiSeqX and NovaSeq and data was compared to whole exome sequencing (WES). To investigate linearity with dilution, specimens from 3 patients were used to make 8 dilutions. Repeatability was investigated using six replicate specimens from each of ten patients, and the effects of cfDNA input were investigated using 1, 5, 20, and 50 ng of cfDNA from seven patients. To investigate the effects of tube type and delayed processing, blood was collected from twenty-three patients into EDTA and Streck BCT tubes and stored for <4 (3 patients), 6-8 (8 patients), or 12-24 h (12 patients) at room temperature before plasma separation.
Summary of Findings:
GC-corrected mean absolute deviation (MAD) scores were very similar between the two sequencing instruments (HiSeqX versus NovaSeq) and the data did not cluster by instrument during hierarchical clustering. Through dilution of three specimens, the authors identified an increase in mean normalized standard deviation (MNSD) of the ULP-WGS tumor fraction when the tumor fraction was <3%. When the tumor fraction was 3 or 4% by ULP-WGS, the sensitivity for mutations detected by WES was 97.2-100% both at 1 x depth and when downsampled to 0.1 x depth. For specimens with a tumor fraction >10% and >5% the MNSDs for replicate specimens were <0.19 and <0.35, respectively; for the three specimens with tumor fractions between 3-5%, the MNSD ranged from 0.33-0.54. Overall, quality metrics (mean depth, library size, or MAD) did not differ between matched specimens that were collected in EDTA and Streck tubes and stored for up to 24 h at room temperature. Tumor fractions were generally comparable between EDTA and Streck specimens; however, there was a 4.75% absolute error between tube types for the three specimens stored <4 h at room temperature, and the absolute error increased to 7.9% for the 12 specimens stored for 12-24 h. Based on these data, the authors concluded that both tubes are acceptable and that EDTA tubes can be stored for ≤8 h at room temperature. The observed tumor fraction was consistent when the input amount was as low as 1 ng DNA, but one specimen failed to achieve the desired library size (>4 x 107), mean depth (>0.25), MAD (<0.20), and coverage per pass filter gigabase (>0.15) when only 1 ng was used. All sequencing metrics were met in all specimens when libraries were constructed from 20 or 50 ng DNA. Based on this, the authors advise using a minimum input amount of 20 ng, but state that 5 ng is acceptable.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck tube
EDTA tube
Storage Storage duration <4 h
6-8 h
12-24 h
Next generation sequencing Specific Template/input amount 1 ng
5 ng
20 ng
50 ng
Biospecimen Aliquots and Components Biospecimen components Tumor fraction 3-5%
Tumor fraction >5%
Tumor fraction >10%
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated