Figure 2.
(A) qPCR is a common method for quantification of nucleic acid with real-time monitoring of the amplification of target of interest (eg, variant sequence shown with red X). Advantages include ubiquitous presence in most clinical laboratories, fast turnaround time, high sample throughput, and broad dynamic range. Disadvantages include limited number of suitable targets/assays available, relative lack of multiplexing ability, need to validate each target/assay individually, potential for false-negative results because of sample impurity, and limited ability to accurately discriminate between very low levels of target as seen in MRD. (B) Rather than performing the PCR reaction in “bulk,” digital PCR partitions the template of interest into individual compartments (top), improving the performance compared with qPCR because of the lower background error rate (lower right), elimination of template competition, and digital result output, allowing absolute quantification (lower left). Lack of deep multiplexing ability and the need to validate each target/assay individually remain limitations. (C) NGS has revolutionized the initial clinical diagnostic evaluation of AML by allowing for simultaneous evaluation of multiple target regions typically selected from those known to be often mutated in AML. NGS is useful for discovery of mutations present in the range from 5% to 100% of a sample (VAF). However, not all variants detected will be pathogenic somatic mutations, and care should be taken to consider the possibility of identification of homozygous or heterozygous germline variants, as well as loss-of-heterozygosity (LOH) events. Variant discovery below a VAF of 5% using panels designed for profiling variants at diagnosis is challenging because of the lack of sensitivity and high false-positive rates. Red asterisks represent low-level variant calls that should be regarded with particular caution as within the range of background error for conventional NGS. (D) NGS for AML MRD performed in recent high-quality research studies has typically included error correction (upper), by incorporation of UMIs, followed by consensus determination of true (red X) variants vs false positives introduced by the technique (green X) and/or bioinformatic approaches to model background error rates at each nucleotide position in those not having a variant and determine the probability that the observed variant is a true positive (red asterisk) (lower). Figure by Erina He, National Institutes of Health Medical Arts.

(A) qPCR is a common method for quantification of nucleic acid with real-time monitoring of the amplification of target of interest (eg, variant sequence shown with red X). Advantages include ubiquitous presence in most clinical laboratories, fast turnaround time, high sample throughput, and broad dynamic range. Disadvantages include limited number of suitable targets/assays available, relative lack of multiplexing ability, need to validate each target/assay individually, potential for false-negative results because of sample impurity, and limited ability to accurately discriminate between very low levels of target as seen in MRD. (B) Rather than performing the PCR reaction in “bulk,” digital PCR partitions the template of interest into individual compartments (top), improving the performance compared with qPCR because of the lower background error rate (lower right), elimination of template competition, and digital result output, allowing absolute quantification (lower left). Lack of deep multiplexing ability and the need to validate each target/assay individually remain limitations. (C) NGS has revolutionized the initial clinical diagnostic evaluation of AML by allowing for simultaneous evaluation of multiple target regions typically selected from those known to be often mutated in AML. NGS is useful for discovery of mutations present in the range from 5% to 100% of a sample (VAF). However, not all variants detected will be pathogenic somatic mutations, and care should be taken to consider the possibility of identification of homozygous or heterozygous germline variants, as well as loss-of-heterozygosity (LOH) events. Variant discovery below a VAF of 5% using panels designed for profiling variants at diagnosis is challenging because of the lack of sensitivity and high false-positive rates. Red asterisks represent low-level variant calls that should be regarded with particular caution as within the range of background error for conventional NGS. (D) NGS for AML MRD performed in recent high-quality research studies has typically included error correction (upper), by incorporation of UMIs, followed by consensus determination of true (red X) variants vs false positives introduced by the technique (green X) and/or bioinformatic approaches to model background error rates at each nucleotide position in those not having a variant and determine the probability that the observed variant is a true positive (red asterisk) (lower). Figure by Erina He, National Institutes of Health Medical Arts.

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