Benchmarking immunoinformatic repertoire assemblies from bulk RNA-seq and PCR-based V(D)J sequencing using PCR-free SMRT RNA sequencing
Abstract
Immune repertoire sequencing (RepSeq) is a crucial immunomonitoring tool, requiring high sensitivity, accuracy, and minimal bias. Although V(D)J mRNA sequencing using 5′ rapid amplification of cDNA ends (RACE-RepSeq) is broadly used, its reliance on PCR may create distortions. Alternatively, in silico repertoire-reconstruction (ISRR) tools, computing bulk RNA-seq data, raise concerns regarding accuracy. We compared RACE-RepSeq and ISRR with an advanced PCR-independent approach based on single-molecule real-time (SMRT) sequencing of full-length transcripts. All three approaches applied to immunoglobulin transcripts yielded consistent repertoire-level metrics, including V/J gene usage, CDR3 features, and diversity indices. However, discrepancies emerged in somatic hypermutation assessment, isotype distribution, and at the individual clone level, including incomplete clone overlap and inconsistent isotype assignment, even among abundant clones. These methodological biases exceeded sampling effects. Overall, our results delineate the limitations of each approach in samples from B cell-rich tissues and support SMRT sequencing as a useful reference for immune repertoire benchmarking.
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BibTeX
@article{genebrier2026benchmarking,
title = {{Benchmarking immunoinformatic repertoire assemblies from bulk RNA-seq and PCR-based V(D)J sequencing using PCR-free SMRT RNA sequencing}},
author = {Steve Genebrier and Samuel Bastos Serra Trinca and Ophélie Dézé and Marie Cornic and Maïwenn Pineau and Karin Tarte and Michel Cogné},
journal = {iScience},
year = {2026},
month = oct,
doi = {10.1016/j.isci.2026.117564},
url = {https://hal.science/hal-05754572v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Benchmarking immunoinformatic repertoire assemblies from bulk RNA-seq and PCR-based V(D)J sequencing using PCR-free SMRT RNA sequencing
AU - Steve Genebrier
AU - Samuel Bastos Serra Trinca
AU - Ophélie Dézé
AU - Marie Cornic
AU - Maïwenn Pineau
AU - Karin Tarte
AU - Michel Cogné
PY - 2026
DA - 2026/10/15
JO - iScience
DO - 10.1016/j.isci.2026.117564
UR - https://hal.science/hal-05754572v1
AB - Immune repertoire sequencing (RepSeq) is a crucial immunomonitoring tool, requiring high sensitivity, accuracy, and minimal bias. Although V(D)J mRNA sequencing using 5′ rapid amplification of cDNA ends (RACE-RepSeq) is broadly used, its reliance on PCR may create distortions. Alternatively, in silico repertoire-reconstruction (ISRR) tools, computing bulk RNA-seq data, raise concerns regarding accuracy. We compared RACE-RepSeq and ISRR with an advanced PCR-independent approach based on single-molecule real-time (SMRT) sequencing of full-length transcripts. All three approaches applied to immunoglobulin transcripts yielded consistent repertoire-level metrics, including V/J gene usage, CDR3 features, and diversity indices. However, discrepancies emerged in somatic hypermutation assessment, isotype distribution, and at the individual clone level, including incomplete clone overlap and inconsistent isotype assignment, even among abundant clones. These methodological biases exceeded sampling effects. Overall, our results delineate the limitations of each approach in samples from B cell-rich tissues and support SMRT sequencing as a useful reference for immune repertoire benchmarking.
SN - 2589-0042
ER -