. 2019 Mar;21(2):330–342. doi: 10.1016/j.jmoldx.2018.10.008

Table 2.

Pitfalls and Technical Considerations Based on Our Experience with the Assay

Pitfalls	Potential reasons	Suggestions and potential solutions
1–2 dominant sequences <2.5% but well above the polyclonal background, >20×	True clone but low tumor content True clone but inefficient PCR because of improper annealing of primers Targeted immune response with dominant specificity	Repeated testing with additional primers or test a separate sample with higher tumor content If similar results but not meeting criteria for clonality, recommend calling dominant clonal sequence and suggest additional testing in other samples to confirm this represents the index clone.
Multiple dominant sequences (same VJ use) after merging—individually do not meet criteria for clonality (>100,000 reads)	Usually related sequences Several single-base changes and clonal drift associated with ongoing SHM Small insertions/deletions in repetitive regions of the genome—technical or biological	Align sequences and establish relationship If closely related, add percentages and reassess Individual clonal sequences will need to be tracked separately in future samples (Supplemental Figures S4–S6)
Multiple dominant sequences (different VJ uses) and low number of total reads <100,000	Low specific template or inefficient PCR because of improper annealing of primers	Repeated testing with higher DNA input and additional primer sets
Multiple dominant sequences of different VJ use, high total reads (>100,000 reads), and absent or low polyclonal background	Multiple populations—consider biclonal and biallelic rearrangements	Review the clonal frequencies and prediction of productive versus nonproductive to help predict if biclonal and biallelic subsets. Biallelic clones: generally same frequency, 1 productive and 1 nonproductive. Biclonal: widely different frequency >10% both productive or nonproductive Interpret in the context of other ancillary studies. If flow shows several populations, consider retesting on segregated populations.
Low read count despite adequate total DNA input	Low specific template or inefficient PCR because of improper annealing of primers	Check B-cell/plasma cell content by histology and flow cytometry. Repeat with multiple primer sets and adjust DNA content to ensure at least 10 ng of DNA is from lymphoid or plasma cell component
Clonal products with no V or J segment (specified as none)	Short V or J segment precludes family assignment or miss-priming events	Align the sequence to IMGT or BLAT and confirm it represents a true rearrangement. Repeat with additional primer sets to confirm If miss-priming event, do not call clonal If true rearrangement but J segment is too short for family name assignment, can call clonal and use the clone for future tracking Cases with low number of reads have several miss-priming events—very short truncated segments or long products with repetitive regions that do not constitute a true rearrangement. Many of these may be automatically filtered out by the Invivoscribe software and would only be seen if data are reanalyzed with other software (Supplemental Figure S6). Cases with more than one miss-priming event should be interpreted with caution—repeat with other primer sets.

Other technical considerations
Establishing the background: In a true polyclonal background, each sequence is generally <0.5% of all of the rearranged reads. In most clonal cases, background starts at sequence rank 3. In cases with ongoing SHM, multiple dominant sequences of same use can be seen and require additional merging. The polyclonal background may be shifted beyond rank 4. For an adequate clonal determination, the polyclonal background should be highly variable. In cases with low read count, multiple clonal signals, and a background with low variability (oligoclonal pattern), recommend retesting with higher DNA input and additional primer sets. Additional merging: Refer to examples in Supplemental Figures S4–S6. In cases with ongoing somatic hypermutation, differences among reads may exceed two bases and may require additional merging for final clonal determination 1. If the top reads share the same use, recommend alignment of the reads to establish relatedness. Sequences with same length and VJ uses are often highly related. Consider adding the percentages for final clonal call. 2. Single insertion or deletion events of more than two bases lead to independent clonal reads of different sizes but same use. If highly related, consider adding percentages to make final clonal call. Despite their relatedness, individual clonal sequences should be tracked independently in subsequent samples.

Other technical considerations

Establishing the background: In a true polyclonal background, each sequence is generally <0.5% of all of the rearranged reads. In most clonal cases, background starts at sequence rank 3. In cases with ongoing SHM, multiple dominant sequences of same use can be seen and require additional merging. The polyclonal background may be shifted beyond rank 4. For an adequate clonal determination, the polyclonal background should be highly variable. In cases with low read count, multiple clonal signals, and a background with low variability (oligoclonal pattern), recommend retesting with higher DNA input and additional primer sets.
Additional merging: Refer to examples in Supplemental Figures S4–S6.
In cases with ongoing somatic hypermutation, differences among reads may exceed two bases and may require additional merging for final clonal determination

1.
If the top reads share the same use, recommend alignment of the reads to establish relatedness. Sequences with same length and VJ uses are often highly related. Consider adding the percentages for final clonal call.
2.
Single insertion or deletion events of more than two bases lead to independent clonal reads of different sizes but same use. If highly related, consider adding percentages to make final clonal call. Despite their relatedness, individual clonal sequences should be tracked independently in subsequent samples.

BLAST, basic local alignment search tool; BLAT, BLAST-like alignment tool; IMGT, international ImMunoGeneTics information system; SHM, somatic hypermutation.