Figure 1.
Figure 1. Outline of amplicon-based NGS-MRD analysis. (A) Primers containing the complementary sequence (green), a 16–base pair (bp) random unique molecular identifier (red), and a so-called common sequence (blue) are designed around a known mutation (red star) covering a nucleotide sequence of 87 to 155 bp. The first PCR is run for 5 cycles, with primers detailed in the graph, and the product is cleaned up and size selected. (B) The second PCR is run for 25 cycles, with primers containing a complementary sequence to the common sequence (blue), the multiplex identifier (MID; orange), and the Illumina adapter (purple), and the product is cleaned up and size selected. (C) Up to 25 samples are pooled and run on a MiSeq sequencing instrument with 251 cycles in both directions. (D) Sequencing reads are demultiplexed by their MIDs, aligned to the target region, and error corrected by reconstructing read families using the random barcodes introduced in the first PCR and by constructing R1/R2 consensus sequences. The x-axis shows nucleotides around the mutated target region; the y-axis shows the variant allele frequency. The left graph (i) shows the graphical representation of the RF analysis. A blue dot shows the largest variant allele frequency (LVAF) at the respective nucleotide position. The black vertical line indicates the base position of the target mutation and in this example shows a mutation clearly above the background sequencing error. The gray vertical lines indicate ±20 bp up- and downstream of the target peak. The black horizontal line indicates the mean background error calculated from LVAFs. The green horizontal line indicates the background error +3 standard deviations of the background error (the target LVAF should be above this line). The red horizontal line indicates the LVAF of the target −3 standard deviations of the background error (no other peak should be above this line within ±20 bp of the target LVAF). The right graph (ii) shows the graphical representation of the R1/R2 corrected analysis. LVAFs are plotted for each nucleotide position. Vertical and horizontal lines are defined as in the left panel. PE, paired end.

Outline of amplicon-based NGS-MRD analysis. (A) Primers containing the complementary sequence (green), a 16–base pair (bp) random unique molecular identifier (red), and a so-called common sequence (blue) are designed around a known mutation (red star) covering a nucleotide sequence of 87 to 155 bp. The first PCR is run for 5 cycles, with primers detailed in the graph, and the product is cleaned up and size selected. (B) The second PCR is run for 25 cycles, with primers containing a complementary sequence to the common sequence (blue), the multiplex identifier (MID; orange), and the Illumina adapter (purple), and the product is cleaned up and size selected. (C) Up to 25 samples are pooled and run on a MiSeq sequencing instrument with 251 cycles in both directions. (D) Sequencing reads are demultiplexed by their MIDs, aligned to the target region, and error corrected by reconstructing read families using the random barcodes introduced in the first PCR and by constructing R1/R2 consensus sequences. The x-axis shows nucleotides around the mutated target region; the y-axis shows the variant allele frequency. The left graph (i) shows the graphical representation of the RF analysis. A blue dot shows the largest variant allele frequency (LVAF) at the respective nucleotide position. The black vertical line indicates the base position of the target mutation and in this example shows a mutation clearly above the background sequencing error. The gray vertical lines indicate ±20 bp up- and downstream of the target peak. The black horizontal line indicates the mean background error calculated from LVAFs. The green horizontal line indicates the background error +3 standard deviations of the background error (the target LVAF should be above this line). The red horizontal line indicates the LVAF of the target −3 standard deviations of the background error (no other peak should be above this line within ±20 bp of the target LVAF). The right graph (ii) shows the graphical representation of the R1/R2 corrected analysis. LVAFs are plotted for each nucleotide position. Vertical and horizontal lines are defined as in the left panel. PE, paired end.

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