Clinical sequencing: From raw data to diagnosis with lifetime value

Abstract

High-throughput sequencing (HTS) has revolutionized genetics by enabling the detection of sequence variants at hitherto unprecedented large scale. Despite these advances, however, there are still remaining challenges in the complete coverage of targeted regions (genes, exome or genome) as well as in HTS data analysis and interpretation. Moreover, it is easy to get overwhelmed by the plethora of available methods and tools for HTS. Here, we review the step-bystep process from the generation of sequence data to molecular diagnosis of Mendelian diseases. Highlighting advantages and limitations, this review addresses the current state of (1) HTS technologies, considering targeted, whole-exome, and whole-genome sequencing on short-and long-read platforms; (2) read alignment, variant calling and interpretation; as well as (3) regulatory issues related to genetic counseling, reimbursement, and data storage.

Hungarian Marfan family with large FBN1 deletion calls attention to copy number variation detection in the current NGS era

“…One of the advantages of an appropriate NGS methodology is that it allows not only the detection of SNVs and small indels but also of CNVs in a single assay. However, not all NGS applications are equally suited for CNV detection (1,16). While PCRfree WGS data facilitates CNV detection and enables CNV characterization at base-pair level due to relatively even read depth and continuous coverage (1,12), the …”

“…As gene panel NGS is restricted to selected amplicons/genes and limited in the detection of deletions and duplications affecting more than 20 bp (1), we subsequently carried out 60× PE150 PCR-free wholegenome sequencing (WGS) on a HiSeq X Ten platform (Illumina, San Diego, CA, USA) as previously described (12). WGS can not only identify sequence variants throughout the genome but has also the advantage of the most continuous coverage, improving both SNV and CNV detections (1).…”

“…Standard NGS analysis pipelines are often restricted to the calling of single nucleotide variants (SNVs) and small insertions/deletions (indels), thereby missing copy number variations (CNVs) including deletions and duplications affecting more than 20 bp (1). However, pathogenic CNVs account for about 10% of disease-causing variants listed in the Human Gene Mutation Database (HGMD Professional 2018.1) and thus should not be missed.…”

Personalized medicine—concepts, technologies, and applications in inflammatory skin diseases

“…landscapes) of biologic pathways, as well as to be able to predict the dynamic effects of perturbations, such as targeting Box 3. Omics technologies for integrative, personalized medicine Genomics Next-generation sequencing (NGS) is key to generating the vast amounts of DNA data for whole-genome sequencing (WGS) and whole-exome sequencing (WES) projects (55). Because the exome only comprises 1.5% (40 Mb) of our genome, it can be sequenced at a deeper coverage (>1009 compared to 309), faster (hours compared to days), and at lower price compared to WGS.…”

Variant filtering, digenic variants, and other challenges in clinical sequencing: a lesson from fibrillinopathies

“…The mother (Ac3) presented with MFS-associated elbow hyperextension, mild scoliosis, and no cardiovascular involvement ( First, although WGS is the most comprehensive HTS method offering advantages over targeted sequencing and WES, 25,35 data interpretation remains a challenge. 12 Currently, there is no generally accepted gold standard software solution for variant interpretation comparable to BWA/GATK for alignment and variant calling. 11,12 According to the ACMG/AMP guidelines, allele frequencies higher than expected for the disorder (criterion BS1) or reported >5% in population-based reference datasets (criterion BA1) count as strong or stand-alone evidence of benign impact of a variant.…”