A novel NGS library preparation method for detection of mutations and gene-fusions in circulating cell-free DNA and RNA


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Guoliang Fu1,Sarah Scaife1,Winnie Wu2
1Genefirst ltd,2genefirst ltd

Abstract

Background

Circulating cell-free tumour DNA- or RNA can be used as a non-invasive biomarker to detect the presence of malignancy, follow treatment response or monitor for recurrence of cancer. Next-Generation Sequencing (NGS) has revolutionised genomic studies and is driving the implementation of precision diagnostics. However, the high sequencing error rate of ∼1% is a fundamental limiting factor when aiming to identify rare mutants in genetically heterogeneous mixtures. Current targeted amplicon-based sequencing techniques are limited in their ability to detect gene fusions/ translocations, and to simultaneously sequence circulating DNA and RNA, especially micro RNA.

Method

To overcome the limitations of current methods, we have developed an improved NGS technology - TBD-seq (tagged bidirectional sequencing)- for the detection of mutations or gene-fusions in DNA and RNA from blood with enhanced sensitivity. A special technique for adding sequencing adaptors into single-stranded fragment ends is used (which does not involve ligation and PCR), to efficiently convert original molecules into sequence ready templates. A unique molecule barcode is introduced to each original molecule which can be used to correct sequencing errors.

Conclusion

We have developed a cancer mutation hotspot panel which includes 3000 mutations of 50 oncogenes and tumour suppressor genes. The library preparation requires as little as 10ng of plasma DNA and takes about five hours to complete. The method demonstrates simplicity, high efficiency and enhanced sensitivity for detecting mutation, gene fusions and RNA.