Next-Generation Sequencing in High-Sensitive Detection of Mutations in Tumors

Abstract

Next-generation sequencing (NGS) technologies have come of age as preferred technologies for screening of genomic variants of pathologic and therapeutic potential. Because of their capability for high-throughput and massively parallel sequencing, they can screen for a variety of genomic changes in multiple samples simultaneously. This has made them platforms of choice for clinical testing of solid tumors and hematological malignancies. Consequently, they are increasingly replacing conventional technologies, such as Sanger sequencing and pyrosequencing, expression arrays, real-time PCR, and fluorescence in situ hybridization methods, for routine molecular testing of tumors. However, one limitation of routinely used NGS technologies is the inability to detect low-level genomic variants with high accuracy. This can be attributed to the frequent occurrence of low-level sequencing errors and artifacts in NGS workflow that need specialized approaches to be identified and eliminated. This review focuses on the origins and nature of these artifacts and recent improvements in the NGS technologies to overcome them to facilitate accurate high-sensitive detection of low-level mutations. Potential applications of high-sensitive NGS in oncology and comparisons with non-NGS technologies of similar capabilities are also summarized.

Recent Advances in the Use of Molecular Analyses to Inform the Diagnosis and Prognosis of Patients with Polycythaemia Vera

“…In recent years, more sensitive detection methods, such as sequencing of plasma RNA [ 25 ] and NGS [ 26 , 27 ], are being increasingly employed for the molecular diagnosis of patients with PV. NGS provides the advantage of potentially detecting all JAK2 variants, including rare and/or previously undescribed mutations [ 26 ].…”

Next Generation Sequencing for Gene Fusion Analysis in Lung Cancer: A Literature Review

“…They found that the use of short amplicons may result in a higher fraction of analyzed DNA in comparison to NGS methods including ligation and clean up. In this study authors reported a median depth of 69.000X, the target depth described for FoundationACT was greater than 5.000X unique median coverage [ 23 ] and the target depth for G360 v2.10 was about 15.000X [ 64 ].…”

“…Template DNA on the beads within each well is sequenced by using unlabeled and unmodified nucleotides, which are introduced in the sequencing reaction one at a time in different flows, according to a precise order. Whenever a nucleotide is incorporated, a hydrogen ion is released causing a pH variation, which is detected by the semiconductor chip, chemical signal is then translated into digital signal [ 23 ]. Illumina technology permits the use of both a hybridization capture-based method and an amplicon-based approach; Ion Torrent applications only use the amplicon-based method [ 13 ].…”

“…Bioinformatics software and analysis algorithms greatly impact on technical sensitivity and specificity [24]; commercially available panels are usually coupled with specific analysis packages, moreover, different innovative analysis methods for gene fusions are continuously being developed [25][26][27]. causing a pH variation, which is detected by the semiconductor chip, chemical signal is then translated into digital signal [23]. Illumina technology permits the use of both a hybridization capture-based method and an amplicon-based approach; Ion Torrent applications only use the amplicon-based method [13].…”

Clinical Utility of Liquid Biopsy-Based Actionable Mutations Detected via ddPCR

“…Although the traditional PCR-based assays are the cheapest ones, they have limited sensitivity and specificity [ 11 ]. Due to the novel mutational status information provided in one single assay by the next-generation sequencing (NGS) technique, their sensitivity and specificity have increased to 0.1% [ 11 , 12 ]. On the downside, NGS is expensive, highly time-consuming, and requires well-experienced bioinformaticians to discriminate between actionable tumoral mutations and normal tissue background [ 13 ].…”