A major challenge in the analytical validation of whole-exome sequensing (WES) assays is the lack of reference exomes for somatic variant calling to establish accurate exome-wide performance characteristics. To address this limitation, we established somatic mutation reference standards using 5 well-characterized cell lines: COLO829, HCC1143, HCC1937, HCC1395, and NCI-H1770.
| Cell line | Diagnosis | Ploidy | TMB | SNV | INDEL | WT genes | Amplifications | Deletions | Neutral |
|---|---|---|---|---|---|---|---|---|---|
| COLO829 | Melanoma | 3 | 6.0 | 489 | 11 | 18'168 | 4'705 | 2'614 | 9'024 |
| HCC1143 | Ductal Carcinoma, Stage IIA | 3 | 5.0 | 379 | 11 | 18'445 | 8'187 | 2'973 | 4'599 |
| HCC1397 | Ductal Carcinoma, Stage IIB | 3 | 5.5 | 405 | 16 | 18'339 | 5'495 | 4'855 | 4'319 |
| HCC1395 | Ductal Carcinima, Stage I | 3 | 15.5 | 1'018 | 55 | 17'374 | 3'341 | 5'414 | 3'750 |
| NCI-H1770 | NSCLC, Stage 4 | 3 | 30.5 | 1'253 | 9 | 15'918 | 2'880 | 7'008 | 6'785 |
Exome-wide somatic reference dataset
Real-world tumor tissues have heterogeneous cell content, it is essential to use representative samples during assay development and validation. To test the assay under challeging conditions with decreasesed malignant cell purity the following dataset is proposed. For each cell line and its corresponding normal B cells, separate FFPE blocks were prepared—one for the tumor cells and one for the matched normal cells. These blocks were stored for 3-4 months, after which DNA extracts were prepared from both the tumor and normal FFPE blocks, as well as from tumor and baseline B cell cultures. The tumor and matched normal DNA extracts were then mixed in various proportions (0:100, 10:90, 20:80, 30:70, 50:50, 25:75, 100:0) before proceeding with library construction procedures.
Preparing cell lines mixtures of different tumor purities
The data generated is available at the SRA repository PRJNA1134786