Measurement of uncertainty for STR allele sizing

General recommendations

Based on the assessments of measurement of uncertainty described below, it is recommended that all expansion-positive results, as well as normal alleles close to the threshold (+/2 repeat units) and close to the sequencing read length (e.g. 49 repeats of trinucleotide repeat unit), if deemed as possibly clinically relevant, are assessed by reviewing STR pileup plots (see STR visualisation), and, if required, an orthogonal test is performed.

For detailed recommendations for each STR loci please refer to NHS England Guidelines for Rare Disease Whole Genome Sequencing & Next Generation Sequencing Panel Interpretation & Reporting.

The accuracy and measurement of uncertainty of STR repeat allele sizing by ExpansionHunter depends on overall allele length.

Alleles shorter than sequencing read length

For alleles shorter than a sequencing read length, i.e. where the whole repeat and some flanking regions on both sides can be spanned by a single sequencing read (i.e. <150bp), the estimate of allele size by ExpansionHunter is very accurate.

In comparison with the allele sizes obtained through standard of care testing for the same samples, 98% of the allele sizes from ExpansionHunter were within +/-2 repeat units of the sizes reported by the standard of care tests, and 85% within +/-1 repeat unit.

In many cases, manual inspection of sequencing reads through visualization software (pileup plots) revealed that ExpansionHunter was likely estimate the allele size more accurately than standard of care tests. In a small number of cases where the detected difference from the standard of care test was >2 repeat units, ExpansionHunter overestimated the allele size, suggesting that it is more likely to produce false positive than false negative calls.

Alleles longer than sequencing read length

When allele length exceeds a sequencing read length, the allele sizing accuracy of ExpansionHunter is reduced.

Validation against standard of care methods showed that clinically relevant STR expansions can still be detected with high sensitivity (see Appendix G for up to date values of STR detection performance), however estimates of exact repeat length are less accurate.

Special notes regarding FMR1

Full FMR1 expansions (>200 repeats) cannot be distinguished from pre-pathogenic expansions and therefore in majority of cases will be reported as pre-expansions in Tier 2.

Tiering of FMR1 was introduced in the NGIS Grace release. For the cases processed with the earlier version of tiering, that was not considering FMR1 despite it being green in PanelApp, a warning will be displayed in the Interpretation Portal notifying that FMR1 was not considered in tiering.

Special notes regarding NOP56

There is a known limitation of ExpansionHunter’s v2.5.6 performance for NOP56, where on some occasions it silently skips genotype calling for this locus when there are no reliably mapped reads due to locus complexity. This leads to missing NOP56 genotypes and therefore the testing for NOP56 should be considered as not performed. Clinical Scientists may want to caveat reports appropriately if there is no results for NOP56 displayed in the Interpretation Portal when they would be expected (i.e. STR locus is green on the panel applied).