The Genomics England 100kGP Transcriptomics Pilot and Extension comprises RNA-sequencing of a subset of rare disease probands from the 100,000 Genomes Project who did not receive a genetic diagnosis through the Genomics England Interpretation Pipeline (7840 samples from 7829 probands: 5546 samples in the initial Pilot project, 2294 samples in the Extension). We prioritised probands who were found to carry variants of unknown significance.

Priorities were based on:

• Variants highlighted through Splice AI
• Autosomal recessive disorders with only a single pathogenic variant identified
• GMC-selected VUS AND contribution to phenotype partial / unknown AND variant type likely to affect RNA processing
• Based on outcome questionnaire and a call to clinicians
• VUS with a high Exomiser score AND variant likely to results in detectable abnormal RNA processing
• Disorder category ranking by Genomics England on the basis of likely monogenic cause (ranks 1-5) for participants from 1.1 AND no diagnosis in outcome questionnaire
• Call to GMCs / clinicians to propose cases based on strong phenotype for a monogenic disorder with no lead from WGS
• Review whether RNA sample is available or requirement for fresh RNA sample