Expansion call by TP-PCR and STR
STR sizing amplifies the repeat with primers in the flanking sequence and sizes both alleles by capillary electrophoresis, which resolves alleles in the normal and premutation range to the single repeat. Repeat-primed PCR anneals one primer inside the repeat, so an expanded allele too long to amplify end to end still produces a ladder of products, and the allele is reported as expanded. The two reactions together classify each allele. DMPK is classed as normal at 34 repeats or fewer, premutation at 35 to 49 and expanded at 50 or more; TCF4 as normal at 39 or fewer and expanded at 40 or more.
Long-range PCR sequencing
Locus-specific primers amplify a fragment of 2,000 to 8,000 bases that spans the whole repeat. Amplicons from many samples carry their own barcodes, are pooled and are read end to end on a nanopore flow cell, so every read spans the repeat and its length gives the repeat length of one amplified copy. Thousands of reads per locus give the length distribution of the amplified allele population and the sequence of any interruptions. The ten-locus panel (DMPK, CNBP, TCF4, HTT, FXN, FMR1, C9orf72, ATXN1, ATXN2 and ATXN3) runs in two multiplexed pools from one 100 ng sample. PCR copies shorter alleles more efficiently than longer ones, so the largest alleles are under-represented in the distribution; every report states this bias.
Small-pool PCR by sequencing
The DNA is diluted until each reaction holds one to five genome equivalents, and 24 to 48 reactions are run per sample, each with its own barcode. At that dilution a reaction amplifies one or a few template molecules, so the set of reactions samples individual alleles from the original DNA, including long alleles that bulk PCR under-represents. The reactions are sequenced together, and the read lengths within each reaction give the alleles it contained. The result is the progenitor allele, the instability index and the single-molecule length distribution defined by the established small-pool method, read by sequencing in place of a Southern blot.
Services and prices
| Service | Reported | Input DNA | Turnaround | Price |
|---|---|---|---|---|
| TP-PCR and STR screen | Both alleles sized by STR in the normal and premutation range; an allele beyond that range is detected by TP-PCR and reported as expanded | 100 ng | 5 business days; rush 3 | $350 per sample |
| Long-range PCR sequencing, one locus | Length distribution of the amplified alleles (mode, median, 10th to 90th percentile) and the interruption sequence | 20 to 100 ng | 5 business days; rush 3 | $250; $150 for each further locus on the same sample |
| Long-range PCR panel, 10 loci | The single-locus report at each of the ten panel loci | 100 ng | 5 business days; rush 3 | $795 per sample |
| Small-pool PCR by sequencing | Progenitor allele, instability index and the single-molecule allele length distribution | 200 ng | 15 business days | $1,495 per sample |
Prices are per sample in US dollars, for research use. Rush turnaround adds 50 percent. The order discounts on the Pricing page apply. Input requirements and shipping are on the Sample requirements page.
PCR sizing and 3X-TGT
| Property | 3X-TGT (no amplification) | PCR sizing |
|---|---|---|
| DNA input | 3 to 15 µg, by read level | 20 to 200 ng |
| DNA quality | Intact high-molecular-weight DNA | Fragments longer than the amplicon; degraded and FFPE-derived DNA accepted |
| Methylation | Reported on every read | Not reported; PCR copies carry no methylation |
| Long alleles | Each molecule is read once, whatever its length | Shorter alleles amplify more efficiently, so the largest alleles are under-represented |
| Typical use | Distribution, interruptions and methylation where DNA is plentiful | Muscle biopsies, mouse tail snips, small swabs and other limited samples |
Deliverables
| Deliverable | Contents |
|---|---|
| Report | The call or distribution for each locus, the read or reaction counts behind it, and the PCR bias statement |
| Per-read table | Long-range and small-pool PCR: one row per read with repeat length, quality and interruptions |
| Reads | The amplicon reads as FASTQ and aligned BAM |
| Fragment-analysis traces | TP-PCR and STR: the capillary electrophoresis traces |
Order or request a quote · [email protected]. Please do not include medical details.