NextStepDx PLUS

Methodology

Genomic DNA was is from the submitted specimen (if cellular material was submitted). DNA is barcoded and enriched for the coding exons of targeted genes using hybrid capture technology. Prepared DNA libraries are then sequenced using a Next Generation Sequencing technology. Following alignment to the human genome reference sequence (assembly GRCh37 / hg19), variants are detected in regions of at least 10x coverage. Specific specimen coverage is provided in the reference section of this report. Variants are interpreted manually using locus specific databases, literature searches, and other molecular biological principles. All the variants with quality score less than 500 (roughly 40X of coverage for a heterozygous variant) will be confirmed by Sanger sequencing. Only variants classified as pathogenic, likely-pathogenic, or unknown significance which are thought to be related to the patient’s phenotype or test indication are reported. When a single pathogenic or likely pathogenic variant is identified in a gene with autosomal-recessive inheritance, the laboratory ensures that 100% of coding sequences of that gene are covered either through NGS or Sanger sequencing technologies. Bioinformatics: Fulgent Germline Pipeline v2019.1 or v2019.2 is used to generate variant calls for this test. The most up to date gene names based on HUGO Gene Nomenclature Committee (https://www.genenames.org) recommendations are used. If the gene name on report does not match that of ordered gene, please contact the laboratory and details can be provided. Result interpretation is based on the collected information available at the time of reporting; additional information may exist in the future which will not be represented. DNA alterations in regulatory regions or deep intronic regions (greater than 20bp from an exon) will not be detected by this test. All sequencing technologies have limitations. There are technical limitations on the ability of DNA sequencing to detect small insertions and deletions. The laboratory uses a sensitive detection algorithm, however these types of alterations are not detected as reliably as single nucleotide variants. Rarely, due to systematic chemical or computational issues, or human error, DNA variants may be missed. Repeat expansions (eg. trinucleotides or hexanucleotides) are not reliably detected by NGS, and pathogenic expansions will not be detected in disease-related genes, even if those genes are sequenced. Unless otherwise indicated, no other assay has been performed to evaluate the submitted specimen for repeat expansions. Although next generation sequencing technologies and the bioinformatics analysis significantly reduce the contribution of pseudogene sequences or other highly-homologous sequences, sometimes, these may still interfere with the technical ability of the assay to identify pathogenic variant alleles in both sequencing and deletion/duplication analyses. Del/dup analysis can identify deletions or duplications of genomic regions which are two or more contiguous exons in size; single exon deletions or duplications will not be routinely detected by this test. When duplications of DNA are identified, if they have not been well-characterized, it is not possible to discern the genomic location or orientation of the duplicated segment, and therefore it is not possible to determine the effect of the duplication. Where deletions are detected, it is not always possible to determine whether the predicted product will remain in-frame or not.

Alternative methodology depending on variant