Mus musculus strain:BALB/c Raw sequence reads

Article: Accurate and predictive antibodyrepertoire profiling by molecularamplification fingerprintingKhan et al. Sci. Adv. 2016; 2 : e1501371High-throughput antibody repertoire sequencing (Ig-seq) provides quantitative molecular information onhumoral immunity. However, Ig-seq is compromised by biases and errors introduced during library preparationand sequencing. By using synthetic antibody spike-in genes, we determined that primer bias from multiplexpolymerase chain reaction (PCR) library preparation resulted in antibody frequencies with only 42 to 62% accuracy.Additionally, Ig-seq errors resulted in antibody diversity measurements being overestimated by up to5000-fold. To rectify this, we developed molecular amplification fingerprinting (MAF), which uses unique molecularidentifier (UID) tagging before and during multiplex PCR amplification, which enabled tagging of transcriptswhile accounting for PCR efficiency. Combined with a bioinformatic pipeline, MAF bias correction led tomeasurements of antibody frequencies with up to 99% accuracy. We also used MAF to correct PCR and sequencingerrors, resulting in enhanced accuracy of full-length antibody diversity measurements, achieving 98 to100% error correction. Using murine MAF-corrected data, we established a quantitative metric of recent clonalexpansion—the intraclonal diversity index—which measures the number of unique transcripts associated withan antibody clone. We used this intraclonal diversity index along with antibody frequencies and somatic hypermutationto build a logistic regression model for prediction of the immunological status of clones. Themodel was able to predict clonal status with high confidence but only when using MAF error and bias correctedIg-seq data. Improved accuracy by MAF provides the potential to greatly advance Ig-seq and its utility in immunologyand biotechnology.