Detection of low-frequency DNA variants by targeted sequencing of the Watson and Crick strands
Authors:
Joshua D Cohen, Christopher Douville, Jonathan C Dudley, Brian J Mog, Maria Popoli, Janine Ptak, Lisa Dobbyn, Natalie Silliman, Joy Schaefer, Jeanne Tie, Peter Gibbs, Cristian Tomasetti, Nickolas Papadopoulos, Kenneth W Kinzler, Bert Vogelstein
Journal:
Nat Biotechnol. 2021 Oct;39(10):1220-1227. doi: 10.1038/s41587-021-00900-z.Epub 2021 May 3.
Abstract:
Identification and quantification of low-frequency mutations remain challenging despite improvements in the baseline error rate of next-generation sequencing technologies. Here, we describe a method, termed SaferSeqS, that addresses these challenges by (1) efficiently introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching target sequences with strand-specific PCR. The method achieves high sensitivity and specificity and detects variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of <5 × 10-7 mutants per base pair (bp). We demonstrate that it can evaluate mutations in a single amplicon or simultaneously in multiple amplicons, assess limited quantities of cell-free DNA with high recovery of both strands and reduce the error rate of existing PCR-based molecular barcoding approaches by >100-fold.
Detection and quantification of rare mutations with massively parallel sequencing
Authors:
Isaac Kinde, Jian Wu, Nick Papadopoulos, Kenneth W Kinzler, Bert Vogelstein
Journal:
The Proceedings of the National Academy of Sciences, 2011 Jun 7;108(23):9530-5.
Abstract:
The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System (“Safe-SeqS”), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant (“supermutants”) only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.
Detection of rare mutations, copy number alterations, and methylation in the same template DNA molecules
Authors:
Yuxuan Wang, Christopher Douville, Joshua D Cohen, Austin Mattox , Sam Curtis, Natalie Silliman, Maria Popoli, Janine Ptak, Lisa Dobbyn, Nadine Nehme, Jonathan C Dudley, Mahmoud Summers, Ming Zhang, Lan T Ho-Pham, Bich N H Tran, Thach S Tran, Tuan V Nguyen, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W Kinzler, Bert Vogelstein
Journal:
The Proceedings of the National Academy of Sciences, August 2023
Abstract:
The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To further increase the sensitivity of such assays with limited amounts of sample, it would be useful to be able to evaluate the same template molecules for all these changes. Here, we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The epigenetic and genetic alterations present in the DNA molecules can then be obtained from the original and copied strands, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung, and colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics.