Table 2. Concerns and expected errors introduced during AIRR-seq workflows and possible controls to detect them.

A typical workflow consists of 5 steps: Sample collection > Extraction > Amplification > Sequencing > Analysis.

Concern	Mechanism(s)	Example of potential controls
Sequence errors	Enzyme errors (RT, DNA polymerase); Sequencing errors	UMIs for bioinformatic error correction; Spike-in controls with defined sequences to evaluate error rates
Sensitivity	Enzymatic inefficiencies (RT or PCR conditions/polymerase); Sample collection size (e.g., cell input number, purity); Sequencing depth	Spike-in controls (synthetic or cellular) at known concentrations
Specificity	Enzyme bias (RT, DNA polymerase); Analysis pipelines (annotation, error correction)	Spike-in controls with defined sequences to identify overall V/D/J gene amplification bias
Detection of contamination	Bench-level cross contamination (sample mixing or PCR contamination) or barcode jumping during sequencing	Unique spike-in (synthetic) for each sample; UDIs for sequencing barcode crosstalk
Sample quality control	Sample collection or nucleic acid purification	Identified by spectroscopy or agarose electrophoresis
Evaluate batch effects	Subtle differences introduced at all stages of the workflow	Spike-in controls (synthetic or cellular); Parallel biological (clonal or complex) sample
Linearity/accuracy of clonotype quantification	Enzymatic inefficiencies (RT or PCR conditions); Analytical error correction	Spike-in controls (synthetic or cellular) at known concentrations
Reproducibility/Batch effects	All stages	Spike-in controls (synthetic or cellular); Parallel biological (clonal or complex) sample; Comparison of replicate amplifications of the same sample; Comparison of sequences generated on the same sample in different sequencing runs
Data processing	Database/annotation limitations; filtering; error correction; collapsing/consensus algorithms	Spike-in controls (synthetic or cellular); Parallel biological (clonal or complex) sample

RT, reverse transcriptase; UMIs, unique molecular identifiers; UDIs, unique dual indices.