MRD Software – Help (FAQ)
MRD Software – Help (FAQ)
Q1：If I run multiple replicates, will the replicate with the highest read frequency or the average of all replicates be displayed by the software?
A1：In version 2.0.1 of the LymphoTrack MRD Software, the average of all replicates is now displayed instead of the replicate with the highest read frequency as in previous versions of the software.
A2：In order to add a Sample you must completely fill out all of the Subject ID fields including the gene target, and at least one sequence to track before clicking Save.
Q3：I entered multiple samples for my Subject but when I click Perform MRD Analysis I get a warning message that says “One or more subjects contains only 1 sample. If you choose to continue with analysis, graphs will not be generated on the summary report. Click cancel if you would like to add samples.”
A3：This warning usually appears because you only have one timepoint entered for the Low Positive Control so the PDF report cannot generate a longitudinal graphs in the Low Positive Control Summary Report. If you have entered multiple samples for your subject you can click “OK” and continue analysis.
A4：During setup, you must add the Low Positive Control using the “Add Low Positive Control” button. If you add Low Positive Control using the “Add Sample” button it will not be DETECTED in any sequence.
Q5：I used Low Positive Control, but on the Subject Sample Report the Low Positive Control Status displays N/A?
A5：Low positive controls are associated with individual samples (timepoints). The Low Positive Control status will always say N/A for a Subject Sample Report because the software is currently unable to associate the Low Positive Control with a specific Subject.
A6：Analysis is qualitative and is intended to determine if a queried sequence is Detected or Not Detected. Estimated read count and frequency are not intended to establish quantification.
A7：Confidence values are based on statistical calculations involving the number of cells and the total read count. DNA Input does not form a linear relationship with confidence values, and can in fact hurt confidence. If more DNA is tested, but the read depth does not increase, it is possible to decrease confidence, though this decrease will be very small.
A8：The LymphoTrack MRD Software v2.0.1 requires at least 5 reads to report MRD as DETECTED. Please refer to A9 for additional detail on how the software handles replicates.
Q9：For clonal cell equivalents, does the software report ‘total’ clonal cell equivalents across all of the replicates or the average value?
A9：For PCR replicates, the cell equivalents that yield positive signal (>5 target reads) are calculated on a per replicate basis, then summed. For any replicate that has fewer than 5 target reads, it has a cell equivalent of zero. For technical replicates (resequencing the same library), all reads are summed before determining positivity and cell equivalents.
A10：For clonal frequency, the total DNA is converted to total cells, then summed across all biological replicates and used as the denominator.
Clonal Frequency = [Est. Clonal Cell Equivalents / Est. Total Cell Equivalents]
A11：Tracking a sequence for MRD requires a high level of sensitivity and specificity. To prevent cross-contamination due to low levels of index misassignment from impacting results, ensure that the highly clonal sample was not run on the same chip or flow cell as any of the follow-up samples assessed for the same clonal sequence. To ensure specificity, verify the clonal sequence is optimal for MRD assessment by searching for the clonal sequence in a *.combined.fastq_unique_reads.tsv file generated using the LymphoTrack Negative Control with the respective LymphoTrack Assay. It is important that the Negative Control sequence data used for this analysis was generated on an NGS run which did not contain any sample with the potential of containing the clonal sequence in question. Please refer to the MRD Analysis is Locus-Sensitive section in the LymphoTrack MRD Software v2.0.1 Instructions for Use for considerations which may aid in the selection of a suitable MRD sequence for tracking.
Q12：Why is there variability in the number of reads across my replicates and should I remove replicates that fall below the target reads per replicate from the Project Planner?
A12：While the LymphoTrack MRD software can combine multiple replicates with variable read counts, one should observe similar read counts across replicates as they originate the same starting material. Significant variability across PCR replicates may be indicative of an upstream processing issue that should be investigated. Based on the performance of each replicate and the LymphoQuant Internal Control (if used) one can determine if a replicate should be excluded.
A13：Loading large amounts of DNA can inadvertently lead to excessive amounts of PCR inhibitors. To determine if this is the cause, verify the purity of the DNA extraction procedure and the DNA concentration process. In some cases, loading less sample DNA may lead to increased amplification efficiency and higher amplicon concentration. In addition, many post-treatment samples tested for MRD may contain depleted lymphocyte populations, which can lead to reduced Ig/TCR template relative to total DNA input. Additional information may also be obtained by comparing the LymphoQuant Internal Control and sample results to the LymphoTrack Low Positive Control and LymphoTrack Negative Control results.
For Research Use Only (RUO). Not intended for diagnostic purposes.