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High-throughput antibiotic repertoire sequencing (Ig-seq) provides quantitative atomic advice on humoral immunity. However, Ig-seq is compromised by biases and errors alien during library alertness and sequencing. By application constructed antibiotic spike-in genes, we bent that album bent from circuitous polymerase alternation acknowledgment (PCR) library alertness resulted in antibiotic frequencies with alone 42 to 62% accuracy. Additionally, Ig-seq errors resulted in antibiotic assortment abstracts actuality abstract by up to 5000-fold. To adjust this, we developed atomic accession fingerprinting (MAF), which uses adapted atomic identifier (UID) tagging afore and during circuitous PCR amplification, which enabled tagging of transcripts while accounting for PCR efficiency. Accumulated with a bioinformatic pipeline, MAF bent alteration led to abstracts of antibiotic frequencies with up to 99% accuracy. We additionally acclimated MAF to actual PCR and sequencing errors, connected in added accurateness of feature antibiotic assortment measurements, accomplishing 98 to 100% absurdity correction. Application murine MAF-corrected data, we accustomed a quantitative metric of contempo clonal expansion—the intraclonal assortment index—which measures the cardinal of adapted transcripts associated with an antibiotic clone. We acclimated this intraclonal assortment base forth with antibiotic frequencies and actual hypermutation to body a logistic corruption archetypal for anticipation of the immunological cachet of clones. The archetypal was able to adumbrate clonal cachet with aerial aplomb but alone back application MAF absurdity and bent adapted Ig-seq data. Bigger accurateness by MAF provides the abeyant to abundantly beforehand Ig-seq and its account in immunology and biotechnology.
Humoral accustomed acknowledgment profiling has progressed abundantly in contempo years attributable to advancements in next-generation sequencing (NGS) technology and bioinformatic appraisal of antibiotic repertoires (1–4). Unlike classical methods for antibiotic appraisal (for example, serum titers), Ig-Seq offers the abeyant to abduction quantitative atomic abstracts of immunological phenomena accompanying to clonal alternative and expansion, such as clonal frequency, diversity, and actual hypermutation. Ig-seq has been acclimated to abode basal questions in B corpuscle development and adverse (5–9), and additionally as an application-focused apparatus for vaccine development (10–13), immunodiagnostic appraisal (10, 14–16), and monoclonal antibiotic engineering (17–23). However, a aloft claiming in advancing Ig-seq is the attendance of errors alien during sample alertness and sequencing, which leads to acutely inaccurate abstracts of antibiotic assortment (24, 25). Added complicating Ig-seq is that the best accepted library bearing methods use circuitous polymerase alternation acknowledgment (PCR) (5, 7, 8, 10, 11, 13, 15, 17–19, 26, 27), wherein ample panels of advanced album sets are acclimated [for example, up to 148 primers for abrasion capricious abounding (VH)-genes (28)] to atone for the aerial assortment of capricious germline genes (V-genes). Differences in alone album ratios, mispriming, and annealing temperatures can aftereffect in ample analytical biases, essentially influencing the administration of antibiotic repertoire clones and V-genes (28–30). Admitting the accession of a 5′ arrangement switching album to mRNA offers a way to amplify V-genes with a accepted distinct album (29, 31), the breadth of 5′ untranslated regions (UTRs) makes it arduous to balance feature VDJ sequences (fig. S1). Added biases from this adjustment may be present because of the poor adeptness of arrangement switching reactions, aboriginal abortion due to RNA accessory structure, and fiber aggression (32–35).
Here, we aboriginal accustomed an access to quantitatively admeasurement errors and biases present in Ig-seq abstracts by application a set of constructed antibiotic standards [complementary DNA (cDNA)] acicular into biological samples (mouse splenic cDNA). Afterward circuitous PCR library alertness and Illumina sequencing [2 × 300 abject pairs (bp) paired-end], we apparent that there was a desperate overestimation (500-fold) of clonal variants (also referred to as clones); we ascertain clonal variants as antibiotic VH sequences with identical V- and J-genes and identical amino acerbic complementarity free regions 3 (CDR3s). Intraclonal variants, authentic actuality as antibiotic sequences with accepted amino acerbic CDR3s but adapted nucleotide sequences in the blow of the V-gene (excluding advanced album bounden regions), were additionally abstract by up to 5000-fold. We added articular that circuitous PCR alien astringent accession biases, connected in alone 42 to 62% accurateness of spike-in clonal frequencies. This award suggests that immunological interpretations of clonal diversity, selection, and accession would be abundantly masked by errors and accession biases.
To afflicted the boundless inaccuracies in Ig-seq, we developed atomic accession fingerprinting (MAF), which consists of stepwise assimilation of adapted atomic identifiers (UIDs). MAF starts with distinct atom tagging of first-strand cDNA during about-face archetype with a reverse-UID (RID), accouterment a adapted tag to anniversary transcript. Notably, MAF continues by tagging anniversary DNA-RID atom during circuitous PCR accession with a forward-UID (FID), whereby overamplified molecules accept added FIDs than underamplified molecules. This fingerprint of accession for anniversary atom accustomed us to apparatus an algorithm to adapt circuitous accession bent effects. With spike-in standards as a reference, MAF bent alteration resulted in up to 99% accurateness of antibiotic clonal frequencies. A multistage MAF absurdity alteration activity resulted in complete (100%) or about complete (98%) alteration of clonal and intraclonal variants, respectively. This led us to authorize a metric for clonal accession based on intraclonal diversity. To authenticate the immunological acceptation of MAF correction, we performed Ig-seq on hyperimmunized and basal mice. Alone with MAF-corrected abstracts was logistic corruption clay able to abstracted Ig-seq abstracts based on accustomed status, breadth anticipation was bedeviled by clonotype (clonal variants with at atomic 80% amino acerbic similarity) frequencies and the intraclonal assortment index.
RNA spike-in standards are acceptable an capital apparatus to appraise errors and bent in sequencing data, the best notable archetype actuality the enactment of the
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