Svetlana Ustyugova

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.

Genome-wide targeted search for human specific and polymorphic L1 integrations

Retroelements (REs) occupy up to 40% of the human genome. Newly integrated REs can change the pattern of expression of pre-existing host genes and therefore might play a significant role in evolution. In particular, human- and primate-specific REs could affect the divergence of the Hominoidea superfamily. A comparative genome-wide analysis of RE sites of integration, neighboring genes, and their regulatory interplay in human and ape genomes would be of help in understanding the impact of REs on evolution and genome regulation. We have developed a technique for the genome-wide comparison of the integrations of transposable elements in genomic DNAs of closely related species. The technique called targeted genome differences analysis (TGDA) is also useful for the detection of deletion/insertion polymorphisms of REs. The technique is based on an enhanced version of subtractive hybridization and does not require preliminary knowledge of the genome sequences under comparison. In this report, we describe its application to the detection and analysis of human specific L1 integrations and their polymorphisms. We obtained a library highly enriched in human-specific L1 insertions and identified 24 such new insertions. Many of these insertions are polymorphic in human populations. The total number of human-specific L1 inserts was estimated to be ~4000. The results suggest that TGDA is a universal method that can be successfully used for the detection of evolutionary and polymorphic markers in any closely related genomes.

Long L1 insertions in human gene introns specifically reduce the content of corresponding primary transcripts

LINE-1 (L1) retrotransposons comprise about 17% of the human genome and include a recently transposed set of Ta-L1 elements that are polymorphic in humans. Although it is widely believed that L1s play an essential role in shaping and functioning of mammalian genomes, the understanding of the impact of L1 insertions on gene expression is far from being comprehensive. Here we compared hnRNA contents for allele pairs of genes heterozygous for Ta-L1 insertions in their introns in human cell lines of various origin. We demonstrated that some Ta-L1 insertions correlated with decreased content of the corresponding hnRNAs. This effect was characteristic of only nearly full-sized L1s and seemed to be tissue specific.

The Evidence for Increased L1 Activity in the Site of Human Adult Brain Neurogenesis

Retroelement activity is a common source of polymorphisms in human genome. The mechanism whereby retroelements contribute to the intraindividual genetic heterogeneity by inserting into the DNA of somatic cells is gaining increasing attention. Brain tissues are suspected to accumulate genetic heterogeneity as a result of the retroelements somatic activity. This study aims to expand our understanding of the role retroelements play in generating somatic mosaicism of neural tissues. Whole-genome Alu and L1 profiling of genomic DNA extracted from the cerebellum, frontal cortex, subventricular zone, dentate gyrus, and the myocardium revealed hundreds of somatic insertions in each of the analyzed tissues. Interestingly, the highest concentration of such insertions was detected in the dentate gyrus—the hotspot of adult neurogenesis. Insertions of retroelements and their activity could produce genetically diverse neuronal subsets, which can be involved in hippocampal-dependent learning and memory.

Cell line fingerprinting using retroelement insertion polymorphism.

Human cell lines are an indispensable tool for functional studies of living entities in their numerous manifestations starting with integral complex systems such as signal pathways and networks, regulation of gene ensembles, epigenetic factors, and finishing with pathological changes and impact of artificially introduced elements, such as various transgenes, on the behavior of the cell. Therefore, it is highly desirable to have reliable cell line identification techniques to make sure that the cell lines to be used in experiments are exactly what is expected. To this end, we developed a set of informative markers based on insertion polymorphism of human retroelements (REs). The set includes 47 pairs of PCR primers corresponding to introns of the human genes with dimorphic LINE1 (L1) and Alu insertions. Using locus-specific PCR assays, we have genotyped 10 human cell lines of various origins. For each of these cell lines, characteristic fingerprints were obtained. An estimated probability that two different cell lines possess the same marker genotype is about 10-18. Therefore, the proposed set of markers provides a reliable tool for cell line identification.

[A tissue-specific decrease in the pre-mRNA level of L1- and alu-containing alleles of human genes].

LINE1 and Alu retroelements occupy approximately 17 and 13% of the human genome, respectively. They include the evolutionarily youngest element groups Ta-L1, AluYa5, and AluYb8, many inserts of which are polymorphic in the Homo sapiens population. Despite the data on the ability of L1 and Alu elements to cause various modifications of the genome, the effects of these retroelements on gene expression have yet not been studied. Using the RT PCR method, we analyzed the pre-mRNA (heterogeneous nuclear RNA) content of allele pairs of four genes in five human cell lines, heterozygous with respect to intronic inserts of L1 and Alu elements. We showed for the first time a tissue-specific decrease in the pre-mRNA content of the gene allele bearing L1 or Alu inserts relative to the other allele of the same gene lacking the retroelement.

Retroposons in modern human genome evolution

The ascertainment of the rates and driving forces of human genome evolution along with the genetic diversity of populations or separate population groups remains a topical problem of fundamental and applied genomics. According to the results of comparative analysis, the most numerous human genome structure peculiarities are connected with the distribution of mobile genetic retroelements—LTR, LINE1, SVA, and Alu repeats. Due to the wide distribution in different genome loci, conversed retropositional activity, and the retroelements’ regulatory potential, let us regard them as one of the significant evolutionary driving forces and the source of human genome variability. In the current review, we summarize published data and recent results of our research aimed at the analysis of the evolutionary impact of the young retroelements group on the function and variability of the human genome. We examine modern approaches of the polygenomic identification of polymorphic retroelements inserts. Using an original Internet resource, we analyze special features of the genomic polymorphic inserts of AluY repeats. We thoroughly characterize the strategy of large-scale functional analysis of polymorphic retroelement inserts. The presented results confirm the hypothesis of the roles of retroelements as active cis regulatory elements that are able to modulate surrounding genes.

A novel approach to identification of somatic retroelements’ insertions in human genome

The activity of retroelements is one of the factors leading to genetic variability of the modern humans. Insertions of retroelements may result in alteration of gene expression and functional diversity between cells. In recent years an increasing amount of data indicating an elevated level of retroelements’ mobilisation in some human and animal tissues has been reported. Therefore, the development of a system for the detection of somatic retroposition events is required. Here we describe a novel approach to the whole-genome identification of somatic retroelement insertions in human genome. The developed approach was applied for the comparisons of somatic mosaicism levels in two tissues of the investigated individual. A total of 3410 insertions of retroelements belonging to AluYa5 subfamily were identified.