Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy

Abstract

During the past five years , new high-throughput DNA sequencing technologies have emerged; these technologies are collectively referred to as next generation sequencing (NGS). By virtue of sequencing clonally amplified DNA templates or single DNA molecules in a massively parallel fashion in a flow cell, NGS provides both qualitative and quantitative sequence data. This combination of information has made NGS the technology of choice for complex genetic analyses that were previously either technically infeasible or cost prohibitive. As a result , NGS has had a fundamental and broad impact on many facets of biomedical research. In contrast , the dissemination of NGS into the clinical diagnostic realm is in its early stages. Though NGS is powerful and can be envisioned to have multiple applications in clinical diagnostics , the technology is currently complex. Successful adoption of NGS into the clinical laboratory will require expertise in both molecular biology techniques and bioinformatics. The current report presents principles that underlie NGS including sequencing library preparation , sequencing chemistries, and an introduction to NGS data analysis. These concepts are subsequently further illustrated by showing representative results from a case study using NGS for targeted resequencing of genes implicated in hypertrophic cardiomyopathy.

Validation of a Next-Generation Sequencing Assay for Clinical Molecular Oncology

“…The hybridized product is amplified (5) and sequenced on an Illumina HiSeq 2000 or Illumina MiSeq instrument (6). Paired-end reads are aligned to the genome (7), PCR duplicates are removed (8), and variant calls are made (9). Variants are annotated and classified by our internally developed CGW application, using publicly available and proprietary databases, and the case is reviewed and interpreted by a clinical genomicist for sign-out in CGW (10).…”

The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories

“…1,2 It has become apparent that NGS platforms will have practical applications in clinical diagnostics and applications, such as detection of EGFR mutations in lung adenocarcinoma, 3 characterizing RAS and methylation pathway alterations in myeloproliferative diseases and myeloid leukemias, [4][5][6][7] or high-resolution, high-throughput human leukocyte antigen genotyping have been developed. 8,9 Thus far, limited data are available on the technical performance of amplicon deep sequencing in a clinical diagnostic setting.…”

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

“…The theoretical value expected for a heterozygous variant is around 50%, this means that 50% of the sequences would contain the variant while for a homozygous variant, is circa 100%. Unfortunately the situation after a postalignment can be far from the theoretical value, in fact the percentage of reads containing the variant for heterozygous can vary from the 20 to 80% while from 60 to 100% for homozygous 16 . Therefore, in order not to miss the key single nucleotide polymorphisms (SNP), it is important to not to discard mutants with less than 100% percentage of reads for the variant.…”