Repeat expansions and read length
More than 50 inherited diseases are caused by repeat expansions, among them myotonic dystrophy, Huntington’s disease, Friedreich ataxia, Fuchs endothelial corneal dystrophy and C9orf72 ALS. Short-read sequencing reads a few hundred bases at a time, and an expanded repeat can run to thousands of bases, so short reads detect an expansion but cannot size it.
Somatic expansion
The repeat keeps expanding in the body after birth, so each patient carries a range of repeat lengths that shifts with age and differs between tissues. The length inherited at conception and the rate of expansion both affect when symptoms begin. A standard clinical test reports whether an expansion is present, or a size class. 3X-TGT reads each molecule end to end without amplification, so one sample gives the full distribution of repeat lengths: its lower edge estimates the inherited length, and its spread shows how far the repeat has expanded, two measures a size class cannot provide.
Enrichment and sequencing
For each target, design software selects an enrichment that leaves the target on one long fragment and cuts the rest of the genome short. The DNA is cut with standard reagents, and size selection keeps the long fragments and removes more than 90 percent of the genome.
The enriched molecules are barcoded, pooled and read end to end on a long-read sequencer. No step amplifies the DNA, so the reads reflect the molecules in the sample, and no PCR, Cas9 or capture probes are used. The method is patent pending.
Measurements on each molecule
| Measurement | Definition |
|---|---|
| Repeat length | Repeat count on every molecule from both alleles |
| Allele length | Repeat count on each copy of the gene, including an estimate of the length inherited at conception |
| Somatic distribution | The spread of repeat lengths across cells, and how it changes over time |
| Interruptions | Variant repeats within the tract, placed on each molecule; in DM1, interruptions are associated with later onset and milder disease |
| Flanking variants | Single-nucleotide variants near the repeat, which assign each molecule to its chromosome where present |
| Methylation | DNA methylation in and around the repeat, read natively on the same molecule |
Methylation calling is in production and is reported on every read.
Read depth and multiplexing
A standard whole genome gives a handful of reads at a disease gene, none long enough to span an expansion. 3X-TGT reads hundreds of molecules per sample across the full expanded allele. Each sample carries its own barcode, so 12 to 24 samples share one flow cell.
Repeat lengths in DM1 blood
Multiplexed Analysis
Validation and regulatory status
The method has been demonstrated at DMPK (DM1) and TCF4 (Fuchs endothelial corneal dystrophy) in blood and saliva. It is offered for research use now. The DMPK and TCF4 assays are in analytical validation in the 3X Genetics CLIA-registered laboratory (CLIA ID 22D2345868); Massachusetts clinical laboratory license pending. Clinical testing is planned for 2027, in that laboratory or through a partner CLIA laboratory. A next-generation enrichment method for deep reads from small amounts of DNA is under development.
To discuss the technology or a sample set, contact Steven Oldenburg, PhD: [email protected]