Biology subjects

Smirnova, A. O.

Publications and source records attributed to Smirnova, A. O..

2 recordsLinked to original sources

bioRxiv2023

Non-canonical D1-D2 rearrangements are bimodal and represent a conservative hidden stage of VDJ recombination in TCR beta locus

T-cell receptor (TCR) diversity is generated by VDJ recombination. The classical course of TCR beta (TRB) chain production starts with D and J segment recombination and finishes with subsequent recombination between the resulting DJ junction and V segment. In this study, we performed deep sequencing of poorly explored incomplete TRBD1 to TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human incomplete TRB DD rearrangements and validated its authenticity by detecting excision circles with RSS (recombination signal sequence) junctions for the first time. The identified rearrangements generated in compliance with the classical 12/23 rule are common for humans, rats, and mice and contain typical VDJ recombination footprints. Detected bimodal distribution of DD junctions indicates two active recombination sites producing long and short DD rearrangements. Unlike long DD rearrangements, the short ones have unusual origin resulting from non-canonical intrachromosomal RSSs junctions formation. Identified DD rearrangements lead to deleting J1 and C1 segments and creating diverse hybrid D segments, which recombine further with J2 and V segments. Resulting functional TRB VDDJ rearrangements are present in the memory T-cells subset proving its participation in antigen recognition.

immunology↗

bioRxiv2021

The use of non-functional clonotypes as a natural spike-in for multiplex PCR bias correction in immune receptor repertoire profiling

High-throughput sequencing of adaptive immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful TCR/BCR repertoire reconstruction and analysis methods have been developed in the past decade. However, detecting and correcting the discrepancy between real and experimentally observed lymphocyte clone frequencies is still challenging. Here we discovered a hallmark anomaly in the ratio between read count and clone count-based frequencies of non-functional clonotypes in multiplex PCR-based immune repertoires. Calculating this anomaly, we formulated a quantitative measure of V- and J-genes frequency bias driven by multiplex PCR during library preparation called Over Amplification Rate (OAR). Based on the OAR concept, we developed an original software for multiplex PCR-specific bias evaluation and correction named iROAR: Immune Repertoire Over Amplification Removal (https://github.com/smiranast/iROAR). The iROAR algorithm was successfully tested on previously published TCR repertoires obtained using both 5 RACE (Rapid Amplification of cDNA Ends)-based and multiplex PCR-based approaches and compared with a biological spike-in-based method for PCR bias evaluation. The developed approach can increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.

immunology↗