Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants.
Author(s): Beck TF, Mullikin JC, Biesecker LG
Publication: Clin Chem, 2016, Vol. 62, Page 647-54
PubMed ID: 26847218 PubMed Review Paper? No
Purpose of Paper
This paper investigated if using Sanger sequencing to validate mutations identified by next-generation sequencing improves mutation discovery.
Conclusion of Paper
All of the 5894 variants identified by NGS except two were confirmed by Sanger sequencing, but additional rounds of Sanger sequencing were necessary to confirm the variant in 17 cases. The authors conclude that unless multiple Sanger sequencing rounds are performed, Sanger sequencing is more likely to refute a true variant than identify a false positive.
Studies
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Study Purpose
This study investigated the concordance between Sanger sequencing and NGS for selected genes using the ClinSeq cohort, which does not require a specific diagnosis but had a preference for individuals with heart disease. DNA was isolated from the blood of patients using the QIAgen kits based on salting out followed by phenol chloroform extraction using Manual Phase Lock Gel extraction kit. Following extraction, next-generation sequencing was performed using a GAIIx or HiSeq 2000 sequencer and data was compared to that obtained by manual inspection of Sanger sequences. Comparisons between the sequencing results were performed for five genes using 684 specimens and for 19 genes using specimens from five individuals. Resequencing was performed by Sanger sequencing in cases of discordance.
Summary of Findings:
Of the 5660 variants identified in the five genes by NGS, all but 19 (13 variants) were confirmed by Sanger sequencing. Resequencing with the same primers confirmed 13 of the variants. Resequencing with additional primers was able to confirm four variants not confirmed using the original primers and 11 variants that were also confirmed after resequencing with the original primers. The remaining two variants which were not detected with either set of primers using Sanger sequencing had low NGS Most Probable Genotype (MPG) scores (4 and 10). When the sequences from 19 genes from five specimens were compared, 714 variants were identified and Sanger sequencing was able to confirm all 234 for which bidirectional data was available. The replication failure rates were considered comparable for the two datasets (19/5660 and 0/234, P=1.000) and the accuracy of NGS compared to Sanger sequencing was 99.965%.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Cardiovascular Disease
- Not specified
Platform:
Analyte Technology Platform DNA Next generation sequencing DNA DNA sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Next generation sequencing Specific Technology platform Sanger sequencing