Next Era Whole Genome Sequencing
Next Era Whole Genome Sequencing
Next Era test represents cutting edge genome profiling and includes:
- Clinical-grade short-read sequencing
- Research-grade Nanopore long-read whole genome sequencing with methylome
- Research-grade long-read transcriptome
- Pharmacogenomics
Turnaround time is 6-12 weeks.
This is performed in a CLIA-certified laboratory with clinical reports reviewed and signed by an ABMGG-certified laboratory geneticist.
Who is this test for?
- Patients with uninformative or inconclusive prior testing
- Patients with complex phenotypes
- Individuals/families seeking comprehensive diagnostic testing
Sequencing & Interpretation
The Alamya Health Next Era test provides short-read (SR) and long-read (LR) sequencing of the entire human genome. Next Era LR sequencing includes DNA methylation profiling.
Clinical SR Genome Sequencing
Our SR genome is sequenced using a NovaSeq 6000 in a CAP/CLIA certified laboratory. The quality control metrics are such that >95% of targeted bases are covered at >15x, >90% of targeted bases are covered at >20x with unique sequencing reads after de-duplication. The minimum acceptable average read depth is 35x. Bioinformatic analysis and clinical reporting are performed at Breakthrough Genomics using an internally developed pipeline.
All disease-causing variants reported in HGMD® and ClinVar as well as all variants with minor allele frequency (MAF) of less than 1% in gnomAD database are considered. Evaluation is focused on coding exons along with previously reported non-coding and splicing pathogenic or likely pathogenic variants reported in HGMD or ClinVar. All pertinent inheritance patterns are considered. In addition, provided family history and clinical information are used to evaluate identified variants.
Three SV calling algorithms are employed (Lumpy, CNVnator, and Manta) that utilize read depth, SNP information, split reads, and reads which map to two different sites in the genome to detect deletions, duplications, insertions and inversions. The overall sensitivity for SNV and Indels is 99.86% and 99.39%, respectively. Sensitivity for detection of insertions (as opposed to duplications) is currently at approximately 20%; as noted below LR sequencing improves this sensitivity and enables detection of balanced translocations. The ability to detect SVs may be impacted by somatic mosaicism. The detection rate for CNVs ≥2 exons is 95%.
Identified SNV/indels and Copy Number Variants (CNVs) are evaluated with respect to their pathogenicity and causality and are classified as pathogenic, likely pathogenic, variant of unknown significance (VUS), likely benign, and benign. Variants that are related to the phenotype of the patient and those that are potentially causative, i.e., classified as pathogenic, likely pathogenic, or variants of uncertain significance (VUS), are reported. Variants of relevance identified by NGS are continuously and individually validated for quality; those variants meeting internal QC criteria (based on extensive validation processes) are not validated by Sanger. All reported CNVs are confirmed by qPCR.
Long-read genome sequencing
Our long-read genome sequencing test is performed on the Oxford Nanopore Technologies PromethION platform. This sequencing assay has the capacity to 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.
SNVs, indels, and CNVs are annotated and prioritized using the ENLITER toolkit from Breakthrough Genomics . ENLITER integrates variant interpretation and annotation with current literature, known genotype-to-phenotype databases (e.g. ClinVar, OMIM, and DECIPHER), population allele frequencies from gnomAD, and in silico scoring metrics (e.g. SpliceAI and CADD).
SVs, repeat expansions, and aberrant allelic methylation are annotated against known disease genes, disease-associated imprinted regions, and well established methylation signatures, and compared to public allele frequency databases (e.g., gnomAD and 1000 Genomes project) as well as an in-house database.
Two types of DNA methylation changes can be found in support of the pathogenicity (cause of the health concern) of certain genetic variants. One type is a methylation alteration that is localized to a specific genomic region. The second type is 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.
Transcriptome
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.
Candidate gene expression, detected splicing isoforms, and biased allelic expression is investigated on a gene-by-gene basis guided by variants identified through Alamya Generation Beyond genome, or to help resolve variants of uncertain significance in patients who have received clinical sequencing. This test may not be able to determine the consequence of candidate variants in genes with a low level of expression in blood.
Pharmacogenomics
Genes for which there are prescribing guidelines (15 genes). The pharmacogenetic variants reported are those that impact commonly prescribed medications.
Test Limitations
Test results are interpreted in the context of clinical findings, family history, and other laboratory data.
- Due to limitations in technology, certain regions may either not be covered or may be poorly covered such that variants cannot be confidently detected.
- Rare polymorphisms might lead to false negative and false positive results.
- Misinterpretation of results might occur if the information provided is inaccurate or incomplete. If results reported do not support the clinical findings, additional testing should be considered.
Samples
Requirements
Whole blood* (1 EDTA tube); 3.0-5.0mL (0.6mL min)
OR
Saliva (Sponge kits and spit kits available)
OR
DNA Isolated from whole blood: 1µg at 100ng/µl in TE, A260/280 = 1.7-2.0µg
Whole blood (1 EDTA tube): 3.0-5.0mL (1.0mL min)
OR
DNA isolated from whole blood: min of 1µg
* Preferred
** Residual post-test DNA (isolated for whole genome sequencing) can be used for the Infinium MethylationEPIC microarray depending on the quantity and quality.
Tube kits are available at no cost and can be sent directly to the patient’s home or the provider’s clinic. Kits can be ordered from our website.
Shipping & Storage
- Samples should be shipped at ambient temperature (18-25ºC) or refrigerated (2-8º).
- Whole blood is stable for 5 days at ambient temperature (18-25ºC) or 7 days refrigerated (2-8ºC). Do not freeze.
- Saliva is stable for 2 years at 2-8ºC. Do not freeze.
- Extracted DNA is stable for 5 years at 2-8ºC. DNA can also be stored frozen at -25ºC to -15ºC.
Shipping Address
Alamya Health
℅ Breakthrough Genomics
2 Hughes, #100
Irvine, CA 92618
Clinical Report
A clinical report is available as are the raw data files (with some limitations).