The LR genome sequencing test uses the Oxford Nanopore Technologies PromethION platform. This sequencing assay can identify single nucleotide variants (SNVs) and small insertions and deletions (indels) in regions of the genome inaccessible by short-read technologies. It also profiles short tandem repeat expansions, structural variants (SVs) such as insertions, inversions, and translocations, and copy number variants (CNVs).

Basecalling, mapping, variant calling, and phasing are performed using the EPI2ME pipeline developed by Oxford Nanopore Technologies. This pipeline leverages the latest production-level bioinformatics tools to generate a comprehensive set of genomic variants. Details of the analysis pipeline can be found at https://github.com/epi2me-labs/wf-human-variation. Briefly, canonical and modified basecalling is performed with Dorado and Remora; alignment is performed with minimap2 against the GRCh38 reference genome; methylated bases are extracted with modbam2bed; SNVs and indels are called with Clair3; SVs are called with Sniffles2; repeat expansions are called by Straglr; and CNVs are called with QDNAseq. 

Two types of informative DNA methylation changes can be found: i) a methylation alteration that is localized to a specific genomic region; and ii) a specific genome-wide pattern of DNA methylation alterations which constitutes a signature characteristic of a specific disorder. It is possible that a DNA methylation change indicative of a genetic disorder is detected without an associated genomic variant; in such instances, further testing might be indicated.

The Oxford Nanopore Technologies PromethION platform assay involves cDNA sequencing from whole blood RNA depleted for hemoglobin mRNA and ribosomal RNAs. Nanopore LRS enables characterization of complete transcripts and facilitates classification of candidate variants with molecular evidence of aberrant splicing, expression, and allele-specific expression. 

Canonical and modified base calling is performed with Dorado. Mapping, transcript assembly, and gene- and transcript-level quantification will be performed using the EPI2ME pipeline developed by Oxford Nanopore Technologies. This pipeline leverages the latest production-level bioinformatics tools to generate a comprehensive view of an individual’s transcriptome. Details of the analysis pipeline can be found at https://github.com/epi2me-labs/wf-transcriptomes. Briefly, FASTQ files are preprocessed with PyChopper, transcript-guided alignment is performed with minimap2 against the GRCh38 reference genome with the Gencode (v43) annotation database, transcript assembly is performed via stringtie, compared against the reference database using gffcompare, and fusion genes are detected by JAFFA.

• This assay is performed for research and has not yet been clinically validated
• Sensitivity for detecting variants varies among genomic regions and variant types such that  the causal variant may not be detected