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1.
PLoS One ; 10(2): e0117854, 2015.
Article in English | MEDLINE | ID: mdl-25689626

ABSTRACT

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.


Subject(s)
Dentate Gyrus/physiology , Long Interspersed Nucleotide Elements/physiology , Neurogenesis/genetics , Neurons/physiology , Brain/physiology , Genome, Human , Humans , Promoter Regions, Genetic
2.
Eur J Immunol ; 42(11): 3073-83, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22806588

ABSTRACT

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.


Subject(s)
Algorithms , High-Throughput Nucleotide Sequencing/instrumentation , High-Throughput Nucleotide Sequencing/methods , Receptors, Antigen, T-Cell/genetics , Sequence Analysis, DNA/methods , Adaptive Immunity/genetics , Adaptive Immunity/immunology , Adult , Base Sequence , Humans , Male , Molecular Sequence Data , Reproducibility of Results , Sequence Analysis, DNA/instrumentation
3.
Genetica ; 128(1-3): 261-72, 2006.
Article in English | MEDLINE | ID: mdl-17028956

ABSTRACT

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.


Subject(s)
Genome, Human , Introns , Long Interspersed Nucleotide Elements , Alleles , Base Sequence , Cell Line , DNA Primers/genetics , Evolution, Molecular , HeLa Cells , Heterozygote , Humans , Jurkat Cells , RNA, Heterogeneous Nuclear/genetics , RNA, Heterogeneous Nuclear/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic
4.
Biotechniques ; 38(4): 561-5, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15884674

ABSTRACT

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.


Subject(s)
Polymorphism, Genetic , Retroelements , Alu Elements , Cell Line , Cell Line, Tumor , DNA Primers , Genetic Markers , HeLa Cells , Humans , Introns , Long Interspersed Nucleotide Elements , Nucleic Acid Amplification Techniques , Polymerase Chain Reaction
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