Technology, which can detect ultra-rare variants that are invisible by other sequencing approaches, was developed by TwinStrand Biosciences. The company is spinoff of The University of Washington. TwinStrand Biosciences is applying this technology to a new generation of high-accuracy clinical diagnostics and innovative tools for scientific discovery.  Current sequencing methods are error-prone, which precludes the identification of low-frequency mutations for early cancer (lat. Carcinoma) detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Duplex Sequencing Technology uses specialized DupSeq to uniquely tag both strands of individual DNA duplexes in a population. This allows identification and comparison of the sequencing read derived from amplified copies of each strand of a particular molecule of origin.

As a rule, errors (red bases) that occur during PCR or sequencing are only present on reads from one strand whereas true mutations (black bases) are present on each strand and are complementary in nature. Duplex Sequencing Error Correction Technology reduces sequencing error rate to below one in ten million and eliminates noise that is inherent in standard Next Generation Sequencing.

Resistance mutation apparent before drug failure using Duplex Sequencing Technology. Duplex Sequencing Technology reveals a low-frequency mutation known to cause imatinib resistance in chronic myeloid leukemia. All other apparent mutations seen with standard DNA sequencing were errors and obscured the presence of this true mutation. Had this mutation been recognized at the time, an alternative, non-cross resistant therapy could have been selected, potentially preventing relapse.

The scientists' aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with high-grade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. They used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, they detected extremely low-frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37).

These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low-frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with the hematologic disorder). Thus, their results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, an age-associated somatic TP53 mutation in noncancerous tissue.

TwinStrand Biosciences has obtained $1,2M of a targeted $4M financing round from undisclosed investors.