Healthcare magazine for research scientists, labs, pathologists, hospitals, cancer centers, physicians and biopharma companies providing news articles, expert interviews and videos about molecular diagnostics in precision medicine
Issue link: https://clinicalomics.epubxp.com/i/853204
www.clinicalomics.com July/August 2017 Clinical OMICs 11 C apillary-based sequencing, also called Sanger sequencing, was once the mainstay of commercial genetic testing. Now, the speed and efficiency of next-genera- tion sequencing (NGS) assays has allowed them to displace Sanger- based tests in most clinical laborator- ies. Yet Sanger sequencing retains a foothold in certain areas, particu- larly the independent confirmation of sequence variants detected by NGS testing. Yet as technologies and analy- sis tools continue to mature, some are asking if even this remains necessary. The Argument against Sanger Confirmation In July 2015, Baudhuin et al. published a study on the necessity of confirm- ing variants in NGS panels by Sanger sequencing. They examined the con- cordance between NGS and Sanger sequencing for 919 NGS variants detected by panel sequencing (177- gene) in 77 samples. Most of these were single nucleotide variants (SNVs) that were required to meet several minimum criteria, including a high variant quality score (>20) and flanking mean base qual- ity (>15), as well as a minimum of 100x sequencing depth. Furthermore, SNVs that mapped near insertion/ deletion variants (indels) or within pseudogenes—arguably the two most significant sources of false positives in next-gen sequencing—were excluded from this comparison. Some 797 SNVs met the above cri- teria, and their overall concordance between NGS and Sanger was 100%. The authors conclude: "Confirmatory analysis by Sanger sequencing of SNVs detected via capture-based NGS testing that meets appropriate quality thresh- olds is unnecessarily redundant." The authors claim- ed 100% concordance for the 122 indels in their comparison, which would be an incredible achieve- ment. However, it's important to note that the authors were primarily confirming coding indels, and their requirements for a match between Sanger and NGS were necessarily less stringent: "For the purposes of this study, if Sanger analysis confirmed the presence of one of the major indels called in a cluster of indel variants reported by NGS, we considered the Sanger and NGS to be concordant for the presence of the indel." I find this problematic, since the size and location of an indel have important implications for its effect on a gene. Perhaps in recognition of this concern the authors admit that "if the genomic location of an indel variant is still uncertain, Sanger confirmatory analysis is indeed appropriate." The Argument for Sanger Confirmation A year later, Ambry Genetics pub- lished a paper examining the results from a slightly smaller (47-gene) hereditary cancer panel, but a much larger sample set: 7,845 sequence vari- ants from 20,000 panel tests that were independently confirmed by Sanger sequencing. Of these, 98.7% were concordant between NGS and Sanger sequencing; 1.3% were identified as NGS false-positives, located mainly in complex genomic regions (A/T-rich regions, G/C-rich regions, homopoly- mer stretches, and pseudogene regions). They conclude that NGS panel tests yield largely accurate results, but still have a false-positive rate above 1%. By adjusting their analysis pipelines, they can get that false-positive rate down to zero, but they lose about 2.2% of true variants. The authors conclude that that Sanger con- firmation is still required for maximum sensitivity in clinical genetic tests. The Balanced Approach: Selective Confirmation of Certain Variants My position is somewhere in the middle. I appreciate that Sanger is a time-consuming and expensive addi- tion to any NGS-based test. This prob- lem will only be exacerbated as we broaden genetic testing to encompass most or all known genes. It's a waste of time and money for the vast major- ity of known variants that meet a mini- mum set of criteria (sequencing depth, quality scores, and possibly variant allele frequency). However, variants that fall short of those criteria, or that have never before been reported (i.e., de novo mutations) should probably be confirmed if they're going on the clin- ical report. That confirmation doesn't need to be Sanger sequencing. In my opinion, a second NGS-based assay using a different enrichment method (e.g. cus- tom capture or digital-droplet PCR) is probably good enough. A version of this column ran previously online at MassGenomics. OP-ED Dan Koboldt Principal Investigator, The Institute for Genomic Medicine, Nationwide Children's Hospital JuliarStudio / Getty Images Sanger Sequencing Still a Necessity in Clinical Labs