ONT rare disease pilot project

Genomics England have sequenced 315 rare disease participants with ONT and have generated structural variant calls. The relevant metadata and paths to files can be found in the rare_disease_ont_cohorts LabKey table.

The participants are grouped in four cohorts and in one of them (cohort_pacbio_pilot) some participants were sequenced more than once using different flow cells:

Cohort name	Cohort description	Number of sequencing runs	Number of participants	Number of families
cohort_CHD	Long read sequencing in familial and syndromic congenital heart disease	92	92	47
cohort_eye	Long read sequencing of rare retinal dystrophies	102	102	99
cohort_pacbio_pilot	Participants selected for PacBio sequencing and with available DNA were sequenced with ONT to enable comparison of different long-read technologies for diagnostic potential. Therefore, the participants in this cohort also appear in the rare_disease_pacbio_pilot table.	128	85	55
cohort_repeat_neuro	Long read sequencing of familial neurological and neurodegenerative disorders	36	36	33

Sequencing information

Sequencing was performed on Oxford Nanopore PromethION 48, using either R9 or R10 flow cells and 4kHz sampling rates. Flow cell types can be found in the flow_cell_product_code column in LabKey:

• FLO-PRO002: R9 flow cell chemistry
• FLO-PRO114M: R10 flow cell chemistry

Bioinformatics tools used to generate files

Base calling was performed with Guppy, using a qscore filter of 7 and either the dna_r9.4.1_450bps_hac_prom or dna_r10.4.1_e8.2_400bps_hac_prom model, depending on flow cell chemistry. Guppy version and base calling models can be found in the basecall_version and basecall_model columns of the Labkey table.

Base called data passing the qscore filter was processed through the GEL R&D long-read pipeline v2.5-2.8 (exact version available in the pipeline_version column). Updates between these pipeline versions are purely operational, each version performs alignment to reference genome GCA_000001405.15_GRCh38_no_alt_analysis_set.fa (available in the RE as /public_data_resources/reference/GRCh38/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna), and structural variant calling with the following software:

• Minimap2: 2.24-r1122
• Sniffles2: 2.0.6

Quality control checks performed

Sequencing QC values were assigned based on the following criteria:

• PASS: Aligned read length N50 >= 5000bps & Total aligned base pairs >= 47,000,000,000 (roughly corresponds to 15X coverage).
• Low_yield: Total aligned base pairs < 47,000,000,000 (roughly corresponds to 15X coverage).
• Low_N50: Aligned read length N50 < 5000bps.

All data is available regardless of their QC value but data sets flagged as low_yield or Low_N50 may not be optimal for variant calling.