Search

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.

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 are 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.

A balanced immune response is a cornerstone of healthy aging. Here, we uncover distinctive features of the long-lived blind mole-rat (Spalax spp.) adaptive immune system, relative to humans and mice. The T-cell repertoire remains diverse throughout the Spalax lifespan, suggesting a paucity of large long-lived clones of effector-memory T cells. Expression of master transcription factors of T-cell differentiation, as well as checkpoint and cytotoxicity genes, remains low as Spalax ages. The thymus shrinks as in mice and humans, while interleukin-7 and interleukin-7 receptor expression remains high, potentially reflecting the sustained homeostasis of naive T cells. With aging, immunoglobulin hypermutation level does not increase and the immunoglobulin-M repertoire remains diverse, suggesting shorter B-cell memory and sustained homeostasis of innate-like B cells. The Spalax adaptive immune system thus appears biased towards sustained functional and receptor diversity over specialized, long-lived effector-memory clones—a unique organizational strategy that potentially underlies this animal’s extraordinary longevity and healthy aging. Investigation of the long-lived blind mole rat adaptive immune system reveals that they do not accumulate large memory T-cell clones and effector programs with aging. Such organization could relieve the burden of inflammaging triggered by persistent clonal expansions and contribute to the long and healthy Spalax lifespan.

Turkey’s involvement in the bombing of Yugoslavia in 1999 was caused by the need to reinforce Turkey’s influence in the Western community, safeguard its regional interests, and support the election campaign. Turkey tried to correlate its policy with that of the West. However, the fundamental interests of Turkey and the West did not coincide. This was “Turkey’s Balkan Dilemma”.

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.

Simple Summary The microalga Haematococcus lacustris is a source of the natural colorant astaxanthin, a powerful antioxidant and key component of cosmetics and animal feed. Haematococcus is cultivated in photobioreactors. It can obtain energy just from a light illuminating photobioreactor and uses inorganic salts and CO2 as sources for chemical elements. The most widespread approach for Haematococcus cultivation is the two stage scheme. At the first stage, biomass accumulation under favorable growth conditions occurs. At the second stage, the cells are subjected to stress inducing astaxanthin synthesis. Generally, the culture of Haematococcus is not axenic. It exists in the form of a community with bacteria constituting its microbiome. The information on photobioreactor-cultivated Haematococcus microbiome is scarce. We analyzed its dynamic during astaxanthin production by DNA metabarcoding and microscopic observations. The main results of the work include the characterization of the daily dynamic of this microbiome and the revealing of contact between microalgae and bacteria. These findings are of potential significance for biotechnology. On one hand, they provide an insight into possible bacterial contamination of the harvested algal biomass. On the other hand, they reveal the presence of a core microbiome or bacteria essential for the growth of the microalga existing in all Haematococcus cultures. Abstract Haematococcus lacustris is a natural source of a valuable ketocarotenoid astaxanthin. Under autotrophic growth conditions, it exists in the form of a community with bacteria. The close coexistence of these microorganisms raises two questions: how broad their diversity is and how they interact with the microalga. Despite the importance these issues, little is known about microorganisms existing in Haematococcus cultures. For the first time, we characterize the dynamic of the H. lacustris microbiome of the microbiome of Haematococcus (a changeover of the bacterial associated species as function of the time) cultivated autotrophically in a photobioreactor based on 16S rRNA metabarcoding data. We found that Proteobacteria and Bacteroidetes are predominant phyla in the community. The Caulobacter bacterium became abundant during astaxanthin accumulation. These data were supported by microscopy. We discuss possible roles and interactions of the community members. These findings are of potential significance for biotechnology. They provide an insight into possible bacterial contamination in algal biomass and reveal the presence of bacteria essential for the algal growth.

Background Retroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare ( Results Here we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions. Conclusions This study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.

The importance of cellular metabolic adaptation in inducing robust T cell responses is well established. However, the mechanism by which T cells link information regarding nutrient supply to clonal expansion and effector function is still enigmatic. Herein, we report that the metabolic sensor adenosine monophosphate‐activated protein kinase (AMPK) is a critical link between cellular energy demand and translational activity and, thus, orchestrates optimal expansion of T cells in vivo. AMPK deficiency did not affect T cell fate decision, activation, or T effector cell generation; however, the magnitude of T cell responses in murine in vivo models of T cell activation was markedly reduced. This impairment was global, as all T helper cell subsets were similarly sensitive to loss of AMPK which resulted in reduced T cell accumulation in peripheral organs and reduced disease severity in pathophysiologically as diverse models as T cell transfer colitis and allergic airway inflammation. T cell receptor repertoire analysis confirmed similar clonotype frequencies in different lymphoid organs, thereby supporting the concept of a quantitative impairment in clonal expansion rather than a skewed qualitative immune response. In line with these findings, in‐depth metabolic analysis revealed a decrease in T cell oxidative metabolism, and gene set enrichment analysis indicated a major reduction in ribosomal biogenesis and mRNA translation in AMPK‐deficient T cells. We, thus, provide evidence that through its interference with these delicate processes, AMPK orchestrates the quantitative, but not the qualitative, manifestation of primary T cell responses in vivo.

COVID-19 is a global pandemic caused by the SARS-CoV-2 coronavirus. T cells play a key role in the adaptive antiviral immune response by killing infected cells and facilitating the selection of virus-specific antibodies. However neither the dynamics and cross-reactivity of the SARS-CoV-2-specific T cell response nor the diversity of resulting immune memory are well understood. In this study we use longitudinal high-throughput T cell receptor (TCR) sequencing to track changes in the T cell repertoire following two mild cases of COVID-19. In both donors we identified CD4+ and CD8+ T cell clones with transient clonal expansion after infection. The antigen specificity of CD8+ TCR sequences to SARS-CoV-2 epitopes was confirmed by both MHC tetramer binding and presence in large database of SARS-CoV-2 epitope-specific TCRs. We describe characteristic motifs in TCR sequences of COVID-19-reactive clones and show preferential occurence of these motifs in publicly available large dataset of repertoires from COVID-19 patients. We show that in both donors the majority of infection-reactive clonotypes acquire memory phenotypes. Certain T cell clones were detected in the memory fraction at the pre-infection timepoint, suggesting participation of pre-existing cross-reactive memory T cells in the immune response to SARS-CoV-2.; COVID-19 is a global pandemic caused by the SARS-CoV-2 coronavirus. T cell response is a critical part of both individual and herd immunity to SARS-CoV-2 and the efficacy of developed vaccines. However neither the dynamics and cross-reactivity of the SARS-CoV-2-specific T cell response nor the diversity of resulting immune memory are well understood. In this study we use longitudinal high-throughput T cell receptor sequencing to track changes in the T cell repertoire following two mild cases of COVID-19 infection. In both donors we identified SARS-CoV-2-responding CD4+ and CD8+ T cell clones. We describe characteristic motifs in TCR sequences of COVID-19-reactive clones, suggesting the existence of immunodominant epitopes. We show that in both donors the majority of infection-reactive clonotypes acquire memory phenotypes. Certain CD4+ clones were detected in the memory fraction at the pre-infection timepoint, suggesting participation of pre-existing cross-reactive memory T cells in the immune response to SARS-CoV-2.

Background and Aims Intestinal inflammation in inflammatory bowel diseases [IBD] is thought to be T cell mediated and therefore dependent on the interaction between the T cell receptor [TCR] and human leukocyte antigen [HLA] proteins expressed on antigen presenting cells. The collection of all TCRs in one individual, known as the TCR repertoire, is characterised by enormous diversity and inter-individual variability. It was shown that healthy monozygotic [MZ] twins are more similar in their TCR repertoire than unrelated individuals. Therefore MZ twins, concordant or discordant for IBD, may be useful to identify disease-related and non-genetic factors in the TCR repertoire which could potentially be used as disease biomarkers. Methods Employing unique molecular barcoding that can distinguish between polymerase chain reaction [PCR] artefacts and true sequence variation, we performed deep TCRα and TCRβ repertoire profiling of the peripheral blood of 28 MZ twin pairs from Denmark and Germany, 24 of whom were discordant and four concordant for IBD. Results We observed disease- and smoking-associated traits such as sharing, diversity and abundance of specific clonotypes in the TCR repertoire of IBD patients, and particularly in patients with active disease, compared with their healthy twins. Conclusions Our findings identified TCR repertoire features specific for smokers and IBD patients, particularly when signs of disease activity were present. These findings are a first step towards the application of TCR repertoire analyses as a valuable tool to characterise inflammatory bowel diseases and to identify potential biomarkers and true disease causes.

The organizational integrity of the adaptive immune system is determined by functionally discrete subsets of CD4+ T cells, but it has remained unclear to what extent lineage choice is influenced by clonotypically expressed T-cell receptors (TCRs). To address this issue, we used a high-throughput approach to profile the αβ TCR repertoires of human naive and effector/memory CD4+ T-cell subsets, irrespective of antigen specificity. Highly conserved physicochemical and recombinatorial features were encoded on a subset-specific basis in the effector/memory compartment. Clonal tracking further identified forbidden and permitted transition pathways, mapping effector/memory subsets related by interconversion or ontogeny. Public sequences were largely confined to particular effector/memory subsets, including regulatory T cells (Tregs), which also displayed hardwired repertoire features in the naive compartment. Accordingly, these cumulative repertoire portraits establish a link between clonotype fate decisions in the complex world of CD4+ T cells and the intrinsic properties of somatically rearranged TCRs.

Significance T cells play a key role in the adaptive immune system. The broad repertoire of unique receptors expressed by T cells is in principle able to recognize a huge diversity of pathogens, but how to extract that information from blood samples remains unclear. By sequencing and analyzing the statistics of T cell receptors of subjects vaccinated against yellow fever, we identified vaccine-specific receptors that expanded following vaccination. We show that each individual has a unique response, which is similar yet across subjects in its sequence composition, with a slightly higher similarity between twins. Our method can be used in the clinic to track disease-specific T cell clones expanding or contracting after infection, vaccination, or therapy., T cell receptor (TCR) repertoire data contain information about infections that could be used in disease diagnostics and vaccine development, but extracting that information remains a major challenge. Here we developed a statistical framework to detect TCR clone proliferation and contraction from longitudinal repertoire data. We applied this framework to data from three pairs of identical twins immunized with the yellow fever vaccine. We identified 600 to 1,700 responding TCRs in each donor and validated them using three independent assays. While the responding TCRs were mostly private, albeit with higher overlap between twins, they could be well-predicted using a classifier based on sequence similarity. Our method can also be applied to samples obtained postinfection, making it suitable for systematic discovery of new infection-specific TCRs in the clinic.

Vaccination against influenza is widely used to protect against seasonal flu epidemic although its effectiveness is debated. Here we performed deep quantitative T cell receptor repertoire profiling in peripheral blood of a healthy volunteer in response to trivalent subunit influenza vaccine. We did not observe significant rebuilding of peripheral blood T cell receptors composition in response to vaccination. However, we found several clonotypes in memory T cell fraction that were undetectable before the vaccination and had a maximum concentration at day 45 after vaccine administration. These cells were found in lower concentration in the course of repertoire monitoring for two years period. Our observation suggests a potential for recruitment of only a limited number of new T cells after each seasonal influenza vaccination.

Background. Genomic rearrangements of T and B cell receptor genes (BCR and TCR) have become the gold standard marker for clonality assessment and minimal residual disease monitoring (MRD) in acute lymphoblastic leukemia (ALL). B-ALL can bear both TCR and BCR rearrangements, whereas T-ALL contains mainly TCR rearrangements. The limitation on the number of potential markers decreases T-ALL cases suitable for MRD monitoring and its reliability. We discovered a missed stage of TRB locus VDJ-recombination during which rearrangement between D1 and D2 segments occurs. Here we examined this new type of rearrangement in pediatric T-ALL as a potential clonal marker for MRD diagnostics for the first time. Methods. Identification of TRB D1-D2 rearrangements was performed using targeted high-throughput sequencing on the Illumina MiSeq instrument [1]. Genomic libraries for sequencing were obtained using PCR with intronic primers flanking the TRBD1 and TRBD2 genes. Genomic DNA for the libraries was extracted from T-ALL patients' bone marrow and peripheral blood of healthy volunteers. DNA from normal CD4+ and CD8+ T-cells was used as a positive control. DNA from normal B-cells and the RMS cell line was used as a negative control. Extraction of TRB D1-D2 rearrangements from sequencing data was performed using modified MiXCR software. Post-analysis was performed using VDJtools software. Results. This study analyzed a cohort of 144 children aged 2-16 years with T-ALL and 12 healthy individuals aged 25-40 years. We identified TRB D1-D2 rearrangements in all analyzed samples except negative controls (B-cells and RMS cell line). The dominant TRB D1-D2 clone in all normal samples occupied 25-30% and had zero length of the D1-D2 junction. The clonal size of the second clone in the top was 3-8%. Based on this observation, we set a 30% cut-off for zero-length D1-D2 junction rearrangements and a 10% cut-off for others to identify abnormally proliferated T-ALL specific clones. We identified 221 different leukemia-related D1-D2 rearrangements in 124 T-ALL samples in total. We intersected them with those in normal samples to test the uniqueness of leukemia-related rearrangements as potential markers. 89% of analyzed rearrangements were specific for leukemia samples, so they can be used for tracking leukemic cells. 11% of rearrangements were present in at least one normal sample and can be used for clonality analysis but are useless for MRD monitoring due to its relatively high generation probability. Zero-length D-D junction was detected as a major clonal marker (25-100%) in 42 T-ALL samples. Nevertheless, 23 out of these 42 samples contained additional unique rearrangements useful for MRD. 29 out of the other 82 samples included one major rearrangement (clonal rate 80-90%), six samples had two major rearrangements with equal clonal rates (30-50%), and the rest of the samples contained 3-5 rearrangements representing oligoclonality. The length of the D-D junction among detected rearrangements in both leukemic and normal samples has a bimodal distribution which is probably linked to two different levels of TdT or exonuclease activity during D1-D2 recombination. Additionally, we observed a strong correlation (Mann-Whitney-U-test, p-value < 0.001) between the presence of TRB D1-D2 rearrangements in T-ALL samples and the CD117 negative immunophenotype of leukemic cells. Conclusions. Obtained results show that TRB D1-D2 rearrangements are a common feature of normal and malignant T-cells. Unique leukemia-related TRB D1-D2 rearrangements can be detected in 73% of pediatric T-ALL cases. It can be routinely detected by PCR with subsequent NGS and can be easily integrated into an existing multiplex system for T-ALL clonality and MRD analysis. References. Komkov et al. High-throughput sequencing of T-cell receptor alpha chain clonal rearrangements at the DNA level in lymphoid malignancies. British Journal of Haematology. 2020 Mar. 188(5): 723-731. Acknowledgment. This work was supported by Russian Science Foundation grant № 20-75-10091 to AK. Disclosures No relevant conflicts of interest to declare.

Migration of Ukrainians to Canada took place in 4 stages: before the first world war, after it, after the second world war and at the end of the XX century. It was caused, respectively, by economic, political, military-political and socio-economic reasons. The official attitude towards Ukrainians in Canada proceeded from racial beliefs about their inferiority compared to the dominant Anglo-Saxons. Although this policy was later transformed into multiculturalism, in reality it was quietly and tacitly applied in a daily practice.

For understanding the rules and laws of adaptive immunity, high‐throughput profiling of T‐cell receptor (TCR) repertoires becomes a powerful tool. The structure of TCR repertoires is instructive even before the antigen specificity of each particular receptor becomes available. It embodies information about the thymic and peripheral selection of T cells; the readiness of an adaptive immunity to withstand new challenges; the character, magnitude and memory of immune responses; and the aetiological and functional proximity of T‐cell subsets. Here, we describe our current analytical approaches for the comparative analysis of murine TCR repertoires, and show several examples of how these approaches can be applied for particular experimental settings. We analyse the efficiency of different metrics used for estimation of repertoire diversity, repertoire overlap, V‐gene and J‐gene segments usage similarity, and amino acid composition of CDR3. We discuss basic differences of these metrics and their advantages and limitations in different experimental models, and we provide guidelines for choosing an efficient way to lead a comparative analysis of TCR repertoires. Applied to the various known and newly developed mouse models, such analysis should allow us to disentangle multiple sophisticated puzzles in adaptive immunity.

The diverse repertoire of T-cell receptors (TCR) plays a key role in the adaptive immune response to infections. Previous studies show that secondary responses to the yellow fever vaccine — the model for acute infection in humans — are weaker than primary ones, but only quantitative measurements can describe the concentration changes and lineage fates for distinct T-cell clones in vivo over time. Using TCR alpha and beta repertoire sequencing for T-cell subsets, as well as single-cell RNAseq and TCRseq, we track the concentrations and phenotypes of individual T-cell clones in response to primary and secondary yellow fever immunization showing their large diversity. We confirm the secondary response is an order of magnitude weaker, albeit 10 days faster than the primary one. Estimating the fraction of the T-cell response directed against the single immunodominant epitope, we identify the sequence features of TCRs that define the high precursor frequency of the two major TCR motifs specific for this particular epitope. We also show the consistency of clonal expansion dynamics between bulk alpha and beta repertoires, using a new methodology to reconstruct alpha-beta pairings from clonal trajectories.

Under unfavorable conditions such as host immune responses and environmental stresses, human pathogen Mycobacterium tuberculosis may acquire the dormancy phenotype characterized by “non-culturability” and a substantial decrease of metabolic activity and global transcription rates. Here, we found that the transition of M. tuberculosis from the dormant “non-culturable” (NC) cells to fully replicating population in vitro occurred not earlier than 7 days after the start of the resuscitation process, with predominant resuscitation over this time interval evidenced by shortening apparent generation time up to 2.8 h at the beginning of resuscitation. The early resuscitation phase was characterized by constant, albeit low, incorporation of radioactive uracil, indicating de novo transcription immediately after the removal of the stress factor, which resulted in significant changes of the M. tuberculosis transcriptional profile already after the first 24 h of resuscitation. This early response included transcriptional upregulation of genes encoding enzymes of fatty acid synthase system type I (FASI) and type II (FASII) responsible for fatty acid/mycolic acid biosynthesis, and regulatory genes, including whiB6 encoding a redox-sensing transcription factor. The second resuscitation phase took place 4 days after the resuscitation onset, i.e., still before the start of active cell division, and included activation of central metabolism genes encoding NADH dehydrogenases, ATP-synthases, and ribosomal proteins. Our results demonstrate, for the first time, that the resuscitation of dormant NC M. tuberculosis is characterized by immediate activation of de novo transcription followed by the upregulation of genes controlling key metabolic pathways and then, cell multiplication.

B-cell receptors and T-cell receptors are the key molecules responsible for specific antigen recognition in adaptive immunity. The huge diversity of immune receptor repertoires constrained their comprehensive studies in the past. More recently, however, high-throughput sequencing based techniques have revolutionized the field of immune receptor repertoire profiling enabling new insights into the development and function of the adaptive immune system. In this review we describe current methods for immune receptor profiling and software tools used for repertoire reconstruction from raw sequencing data. We also provide examples of how immune repertoire profiling can be used to study adaptive immunity in disease and in the course of organ and bone marrow transplantation.

αβT-cell-depleted allogeneic hematopoietic cell transplantation holds promise for the safe and accessible therapy of both malignant and non-malignant blood disorders. Here we employed molecular barcoding normalized T-cell receptor (TCR) profiling to quantitatively track T-cell immune reconstitution after TCRαβ-/CD19-depleted transplantation in children. We demonstrate that seemingly early reconstitution of αβT-cell counts 2 months after transplantation is based on only several hundred rapidly expanded clones originating from non-depleted graft cells. In further months, frequency of these hyperexpanded clones declines, and after 1 year the observed T-cell counts and TCRβ diversity are mostly provided by the newly produced T cells. We also demonstrate that high TCRβ diversity at day 60 observed for some of the patients is determined by recipient T cells and intrathymic progenitors that survived conditioning regimen. Our results indicate that further efforts on optimization of TCRαβ-/CD19-depleted transplantation protocols should be directed toward providing more efficient T-cell defense in the first months after transplantation.

High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d.

Fitness landscapes1,2, depictions of how genotypes manifest at the phenotypic level, form the basis for our understanding of many areas of biology2–7 yet their properties remain elusive. Studies addressing this issue often consider specific genes and their function as proxy for fitness2,4, experimentally assessing the impact on function of single mutations and their combinations in a specific sequence2,5,8–15 or in different sequences2,3,5,16–18. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here, we chart an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function, fluorescence, of tens of thousands of derivative genotypes of avGFP. We find that its fitness landscape is narrow, with half of genotypes with two mutations showing reduced fluorescence and half of genotypes with five mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations arising mostly through the cumulative impact of slightly deleterious mutations causing a threshold-like decrease of protein stability and concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for a number of fields including molecular evolution, population genetics and protein design.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant neoplasm of the lymphocyte precursors that suffered malignant transformation arresting the lymphoid cell differentiation. Clinical studies revealed monoor, more rarely, oligoclonal nature of the disease. A precise identification of malignant clone markers is both the crucial stage of early diagnostics and the essential prognostic factor for therapeutic treatment. Here we present an improved system for unbiased detection of lymphoblastic clones in bone marrow aspirates of T-ALL patients. The system based on multiplex PCR of rearranged T-cell receptor locus (TRB) and straightforward sequencing of the resulted PCR fragments. Testing of the system on genomic DNA from Jurkat cell line and four clinical bone marrow aspirates revealed a set of unique TRB rearrangements that precisely characterize each of tested samples. Therefore, the outcome of the system produces highly informative molecular genetic markers for further monitoring of minimal residual disease in T-ALL patients.

Cancer remains one of the main causes of human mortality despite significant progress in its diagnostics and therapy achieved in the past decade. Massive hypomethylation of retrotransposons, in particular LINE-1, is considered a hallmark of most malignant transformations as it results in the reactivation of retroelements and subsequent genomic instability. Accumulating data on LINE-1 aberrant methylation in different tumor types indicates its significant role in cancer initiation and progression. However, direct evidence that LINE-1 activation can be used as a cancer biomarker is still limited. The objective of this review was to critically evaluate the published results regarding the diagnostic/prognostic potential of the LINE-1 methylation status in cancer. Our analysis indicates that LINE-1 hypomethylation is a promising candidate biomarker of cancer development, which, however, needs validation in both clinical and laboratory studies to confirm its applicability to different cancer types and/or stages. As LINE-1 is present in multiple cell-free copies in blood, it has advantages over single-copy genes regarding perspectives of using its methylation status as an epigenetic cancer biomarker for cell-free DNA liquid biopsy.

Phosphorus (P) is central to storing and transferring energy and information in living cells, including those of microalgae. Many microalgal species dwelling in low P environments are naturally equipped to take up and store P whenever it becomes available through a complex phenomenon known as “luxury P uptake.” Its research is required for better understanding of the nutrient geochemical cycles in aquatic environments but also for biotechnological applications such as sequestration of nutrients from wastewater and production of algal fertilizers. Here, we report on our recent insights into luxury P uptake and polyphosphate formation originating from physiological, ultrastructural, and transcriptomic evidence. The cultures pre-starved of P and re-fed with inorganic phosphate (Pi) exhibited a bi-phasic kinetics of Pi uptake comprising fast (1–2 h after re-feeding) and slow (1–3 d after re-feeding) phases. The rate of Pi uptake in the fast phase was ca. 10 times higher than in the slow phase with an opposite trend shown for the cell division rate. The transient peak of polyphosphate accumulation was determined 2–4 h after re-feeding and coincided with the period of slow cell division and fast Pi uptake. In this phase, the microalgal cells reached the highest P content (up to 5% of dry cell weight). The P re-feeding also reversed the characteristic changes in cell lipids induced by P starvation, namely increase in the major membrane glycolipid (DGDG/MGDG) ratio and betaine lipids. These changes were reversed upon Pi re-feeding of the starved culture. Electron microscopy revealed the ordered organization of vacuolar polyphosphate indicative of the possible involvement of an enzyme (complex) in their synthesis. A candidate gene encoding a protein similar to the vacuolar transport chaperone (VTC) protein, featuring an expression pattern corresponding to polyphosphate accumulation, was revealed. Implications of the findings for efficient biocapture of phosphorus are discussed.

Background There is increasing evidence that the transpositional activity of retroelements (REs) is not limited to germ line cells, but often occurs in tumor and normal somatic cells. Somatic transpositions were found in several human tissues and are especially typical for the brain. Several computational and experimental approaches for detection of somatic retroelement insertions was developed in the past few years. These approaches were successfully applied to detect somatic insertions in clonally expanded tumor cells. At the same time, identification of somatic insertions presented in small proportion of cells, such as neurons, remains a considerable challenge. Results In this study, we developed a normalization procedure for library enrichment by DNA sequences corresponding to rare somatic RE insertions. Two rounds of normalization increased the number of fragments adjacent to somatic REs in the sequenced sample by more than 26-fold, and the number of identified somatic REs was increased by 8-fold. Conclusions The developed technique can be used in combination with vast majority of modern RE identification approaches and can dramatically increase their capacity to detect rare somatic RE insertions in different types of cells. Electronic supplementary material The online version of this article (10.1186/s13100-018-0136-1) contains supplementary material, which is available to authorized users.

BackgroundThere is increasing evidence that the transpositional activity of retroelements (REs) is not limited to germ line cells, but often occurs in tumor and normal somatic cells. Somatic transpositions were found in several human tissues and are especially typical for the brain. Several computational and experimental approaches for detection of somatic retroelement insertions was developed in the past few years. These approaches were successfully applied to detect somatic insertions in clonally expanded tumor cells. At the same time, identification of somatic insertions presented in small proportion of cells, such as neurons, remains a considerable challenge.ResultsIn this study, we developed a normalization procedure for library enrichment by DNA sequences corresponding to rare somatic RE insertions. Two rounds of normalization increased the number of fragments adjacent to somatic REs in the sequenced sample by more than 26-fold, and the number of identified somatic REs was increased by 7.9-fold.ConclusionsThe developed technique can be used in combination with vast majority of modern RE identification approaches and can dramatically increase their capacity to detect rare somatic RE insertions in different types of cells.

Numerous studies have demonstrated that the percentage of naïve T cells and diversity of T cell receptor (TCR) repertoire decrease with age, with some findings likewise suggesting that increased repertoire diversity may be associated with longer lifespan and healthy aging. In this work, we have analyzed peripheral TCR diversity from humans, mice, and blind mole-rats (Spalax spp.)—long-lived, hypoxia- and cancer-tolerant rodents. We employed a quantitative approach to TCR repertoire profiling based on 5’RACE with unique molecular identifiers (UMI) to achieve accurate comparison of repertoire diversity, which also required development of specific wet lab protocol and TCR gene reference for Spalax. Our direct comparison reveals a striking phenomenon. Whereas TCR diversity of mice and humans decreases with age, resulting primarily from the shrinkage of the naive T cell pool, Spalax TCR diversity remains stable even for the animals that reach extreme old age (15-17 years). This indicates that T cell immunity does not meaningfully age in long-lived rodents, at least in terms of the classical understanding of immunosenescence, which is associated with the accumulation of large numbers of memory clones. We suggest that the extraordinary longevity of Spalax may be attributable at least in part to the distinctive organization of their T cell immunity. Our findings should therefore encourage a close re-examination of the contribution of immunosenescence to life span in mammals.

Detection of minimal residual disease (MRD) is a powerful prognostic tool in many hematological malignancies including ALL. Several groups of markers are widely used to monitor the concentration of a malignant clone including the detection of 'clonal B-cell (BCR) or T-cell (TCR) gene receptor rearrangements in ALL. Identification of a clonal rearrangement specific for the malignant clone, which usually constitutes from 20 to 90% in the initial bone marrow sample is a relatively straightforward task. Tracking this rearrangement after therapy is more tricky as the concentration of malignant cells in the sample can be very low. Recent progress in MRD detection based on quantitative real-time PCR (qPCR) and highthroughput sequen- cing (HTS) allows to detect malignant clones present at a concentration of one per 10 and even per 10 cells. Although MRD detection is claimed to be able to accurately predict the outcomes of leukemia therapy in different clinical settings, the value of this information is sometimes corrupted by false-positive and false-negative results of MRD measurement. One of the potential causes of false-positive results is discussed below.