G4 Sequencing Platform on a Solid Tumor Gene Fusion NGS Assay

About Myself

• Faqiang Wu, PhD
• Staff Scientist in Clinical Diagnostics at NeoGenomics Laboratories
• Job responsibility: NGS assay development for cancer testing
• Personal interest: Clinical data analysis and visualization

About NeoGenomics Laboratories

• NeoGenomics, Inc. specializes in cancer genetics testing and information services.
• Providing one of the most comprehensive oncology-focused testing menus in the world for physicians to help them diagnose and treat cancer.
• Serving pharmaceutical clients in clinical trials and drug development.
• CAP accredited and CLIA certified laboratories for full-service sample processing in FL, CA, NC, TX and the United Kingdom.
• Small, non-processing laboratory locations across the United States for providing analysis services.

AMP Poster

Abstract # 1874602 | Poster # TT004

INTRODUCTION

In this study, we evaluated the performance of the G4 sequencing platform developed by Singular Genomics, using a capture-based gene fusion NGS assay from NeoGenomics Laboratories.

Singular’s G4 Sequencing Platform

Figure 1: The G4 Sequencer from Singular Genomics Systems, Inc.

A benchtop sequencing platform delivering accuracy, speed, power, and unprecedented flexibility for a wide range of genomic applications.

Table 1: Specification of G4 sequencer.

Instrument Specifications	Singular G4
SBS Chemistry	4 color
No. of Flow Cells / Instrument	4
No. of Lanes / Flow Cell	4
Bases ≥ Q30	> 85%

Table 2: Specification of F2 and F3 flow cell.

Flow Cell Specifications	F2	F3
Paired Reads / Flow Cell	200 M	400 M
Paired Reads / Run	800M	1.6B
Run time (300 cycles)	~19 hrs	~24 hrs

Targeted Solid Tumor NGS Fusion Panel

• 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.

EXPERIMENT DESIGN

1. Four clinical samples were selected for evaluation.
2. Each sample carries gene fusions previously detected on Illumina NovaSeq 6000 using the Solid Tumor Gene Fusion NGS Assay developed by Genomics Laboratories.
3. When converting the libraries for sequencing on G4, additional PCR cycles were applied to replace Illumina’s P5 and P7 after library capturing.
4. The converted libraries were sequenced on single lanes of F2 flow cells on the Singular G4 sequencer.
5. The sequencing results were run on a modified pipeline for QC and fusion calls.

Figure 2: (A) Workflow of NeoGenomics Solid Tumor NGS Fusion assay. (B) Main difference between the standard workflow (as control) and the modified workflow (for evaluation).

RESULTS

FastQC

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

Figure 3: Representative FastQC results of sample Patient D. Quality scores across all based of Read 1 (A) and Read 2 (B). Quality score distribution over all sequences of Read 1 (C) and Read 2 (D).

Library QC

• 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.

Figure 4: Libary QC of the four patient samples sequenced on Illumina NovaSeq 6000 and Singular G4.

Fusion Call

• 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.

Table 3: List of fusion calls on NovaSeq 6000 and G4

	Fusion genes	Detected on		Concordance
		Illumina NovaSeq 6000	Singular G4
Patient A	WWTR1:CAMTA1	✓	✓	Yes
Patient B	NPTN:HMGN2P46	✓	✓	Yes
	SLC45A3:ELK4	✓	✓	Yes
	TMPRSS2:ERG	✓	✓	Yes
	YWHAE:HAP1	✓	✓	Yes
Patient C	ETV6:NTRK3	✓	✓	Yes
Patient D	APLP2:ST14	✓	✓	Yes
	ARMC8:MBNL1	✓	✓	Yes
	ATE1:FGFR2	✓	✓	Yes
	CYSTM1:ANKHD1	✓	✓	Yes
	GRTP1:DNAJC14		✓	No
	PDGFRA:AL139811.2		✓	No
	PPP2R2A:BNIP3L	✓	✓	Yes
	PXDC1:KPNA4	✓	✓	Yes
	SRP9:DNAH14	✓	✓	Yes

CONCLUSION

• This evaluation study demonstrates that the G4 sequencing platform’s high-fidelity performance matches that of the Illumina platform when applied to a solid tumor gene fusion NGS assay.

• The G4 platform offers a reliable and efficient alternative for genomic research and clinical applications.

ACKNOWLEDGEMENT

NeoGenomics Laboratories:

• Faqiang Wu,
• Long Vu,
• Steven Rivera,
• Segun Jung,
• Yanglong Mou,
• Brad Thomas,
• Cynthie Wong,
• Jiannan Guo
• and more…

Singular Genomics:

• Jordan Williams,
• Jonathan Slasinski,
• Ryan Shultzaberger,
• Laure Moller,
• Eli Glezer,
• Martín Fabani,
• Jeremy Schurman,
• and more…