• A RNA capture-based next-generation sequencing test that detects translocations and fusions with known and novel fusion partners in 250 genes (including EGFRvIII and METex14 variants).

• It detects gene fusions across multiple solid tumors, including lung, brain, breast, thyroid, salivary gland, prostate, sarcoma, colorectal, cholangiocarcinoma, and pancreas.

• Four clinical patient samples were selected for this evaluation study.
• Each sample carries gene fusions previously detected on Illumina NovaSeq 6000 using the Solid Tumor Gene Fusion NGS Assay developed by Genomics Laboratories.

• G4 sequencer from Singular Genomics (F2 flow cells).
• NovaSeq 6000 from Illumina (as control).

1. Four clinical samples with known gene fusions were selected for evaluation.
2. Additional PCR cycles were applied to replace Illumina’s P5 and P7 after library capturing.
3. The converted libraries were sequenced on single lanes of F2 flow cells on the Singular G4 sequencer.
4. The sequencing results were run on a modified pipeline for QC and fusion calls (Fig. 2B).

Quality score of the majority reads are above 30 throughout the cycles, suggesting excellent sequencing quality of Singular G4 sequencer.

• In general, the sequencing quality of G4 is comparable to that of NovaSeq 6000.
• The higher duplicaton in G4 is likely due to the higher initial reads.
• The extra PCR step to replace Illumina flow cell binding sequences does not seem to impact library quality. No obvious differences observed between 5- and 7-cycle PCR.

• All (14/14) fusion genes detected on Novaseq 6000 were detected on G4.
• The additional fusion genes detected on G4 are probably due to the higher sequencing depth on G4 than on NovaSeq 6000.