Next generation sequencing for TCR repertoire profiling: platform-specific features and correction algorithms.

The TCR repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform-specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of next generation sequencing applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.

Bolotin DA ，Mamedov IZ ，Britanova OV ，Zvyagin IV ，Shagin D ，Ustyugova SV ，Turchaninova MA ，Lukyanov S ，Lebedev YB ，Chudakov DM ... -  《-》

TCRklass: a new K-string-based algorithm for human and mouse TCR repertoire characterization.

The next-generation sequencing technology has promoted the study on human TCR repertoire, which is essential for the adaptive immunity. To decipher the complexity of TCR repertoire, we developed an integrated pipeline, TCRklass, using K-string-based algorithm that has significantly improved the accuracy and performance over existing tools. We tested TCRklass using manually curated short read datasets in comparison with in silico datasets; it showed higher precision and recall rates on CDR3 identification. We applied TCRklass on large datasets of two human and three mouse TCR repertoires; it demonstrated higher reliability on CDR3 identification and much less biased V/J profiling, which are the two components contributing the diversity of the repertoire. Because of the sequencing cost, short paired-end reads generated by next-generation sequencing technology are and will remain the main source of data, and we believe that the TCRklass is a useful and reliable toolkit for TCR repertoire analysis.

Yang X ，Liu D ，Lv N ，Zhao F ，Liu F ，Zou J ，Chen Y ，Xiao X ，Wu J ，Liu P ，Gao J ，Hu Y ，Shi Y ，Liu J ，Zhang R ，Chen C ，Ma J ，Gao GF ，Zhu B ... -  《-》

Probing the T-cell receptor repertoire with deep sequencing.

Miconnet I 《-》

Wrestling with the repertoire: the promise and perils of next generation sequencing for antigen receptors.

Next generation sequencing technologies are revolutionizing the study of immune repertoires. These methods provide a previously unimaginable amount of sequence data, unfortunately accompanied by numerous challenges associated with error correction and interpretation that remain to be solved. For antigen receptors, these challenges will require dedicated solutions beyond those developed for genome sequencing, which may differ depending on the sequencing technology used and the purpose of the experiment. Many investigators are developing such methods, based on different sequencing platforms, but critical details of protocol and performance are proprietary. The field will move forward when these methods are shared and standardized, and when the accuracy, sensitivity and reproducibility of various sequencing, and analytic methods are evaluated using standardized samples in comparative experiments.

Baum PD ，Venturi V ，Price DA 《-》

Clinical T Cell Receptor Repertoire Deep Sequencing and Analysis: An Application to Monitor Immune Reconstitution Following Cord Blood Transplantation.

Gkazi AS ，Margetts BK ，Attenborough T ，Mhaldien L ，Standing JF ，Oakes T ，Heather JM ，Booth J ，Pasquet M ，Chiesa R ，Veys P ，Klein N ，Chain B ，Callard R ，Adams SP ... -  《Frontiers in Immunology》