From Sanger to genome sequencing - an overview of DNA sequencing technologies / Od Sangera do sekwencjonowania genomów ­ przeglad technologii sekwencjonowania DNA.

There is no technique that would make a greater contribution to the development of genetics, molecular biology and medicine than DNA sequencing. For many years, the method based on enzymatic DNA synthesis developed by Frederic Sanger was the gold standard in this area. At the end of the 20th century, there was a dynamic development of next-generation sequencing (NGS) technologies, which ended the era of single gene analysis and initiated the era of genome sequencing. Despite fierce competition, one NGS technology has practically completely dominated the global market. In the article, we present our own review of DNA sequencing methods, starting from the Sanger method to high-throughput second- and third-generation sequencing technologies, with particular emphasis on those that have achieved commercial success. We present their short history, principles of operation, technical possibilities, applications and limitations. In the summary, we reveal how much human genome sequencing costs at the current stage of the genomic revolution and outline the prospects for further development of genomics.

The landscape of the COVID-19 pandemic in Poland emerging from epidemiological and genomic data.

The COVID-19 pandemic has profoundly affected all aspects of our lives. Through real-time monitoring and rapid vaccine implementation, we succeeded in suppressing the spread of the disease and mitigating its consequences. Finally, conclusions can be summarized and drawn. Here, we use the example of Poland, which was seriously affected by the pandemic. Compared to other countries, Poland has not achieved impressive results in either testing or vaccination, which may explain its high mortality (case fatality rate, CFR 1.94%). Through retrospective analysis of data collected by the COVID-19 Data Portal Poland, we found significant regional differences in the number of tests performed, number of cases detected, number of COVID-19-related deaths, and vaccination rates. The Masovian, Greater Poland, and Pomeranian voivodeships, the country's leaders in vaccination, reported high case numbers but low death rates. In contrast, the voivodeships in the eastern and southern parts of Poland (Subcarpathian, Podlaskie, Lublin, Opole), which documented low vaccination levels and low case numbers, had higher COVID-19-related mortality rates. The strong negative correlation between the CFR and the percentage of the population that was vaccinated in Poland supports the validity of vaccination. To gain insight into virus evolution, we sequenced more than 500 genomes and analyzed nearly 80 thousand SARS-CoV-2 genome sequences deposited in GISAID by Polish diagnostic centers. We showed that the SARS-CoV-2 variant distribution over time in Poland reflected that in Europe. Haplotype network analysis allowed us to follow the virus transmission routes and identify potential superspreaders in each pandemic wave.

Genetic history of East-Central Europe in the first millennium CE.

BACKGROUND: The appearance of Slavs in East-Central Europe has been the subject of an over 200-year debate driven by two conflicting hypotheses. The first assumes that Slavs came to the territory of contemporary Poland no earlier than the sixth century CE; the second postulates that they already inhabited this region in the Iron Age (IA). Testing either hypothesis is not trivial given that cremation of the dead was the prevailing custom in Central Europe from the late Bronze Age until the Middle Ages (MA). RESULTS: To address this problem, we determined the genetic makeup of representatives of the IA Wielbark- and MA Slav-associated cultures from the territory of present-day Poland. The study involved 474 individuals buried in 27 cemeteries. For 197 of them, genome-wide data were obtained. We found close genetic affinities between the IA Wielbark culture-associated individuals and contemporary to them and older northern European populations. Further, we observed that the IA individuals had genetic components which were indispensable to model the MA population. CONCLUSIONS: The collected data suggest that the Wielbark culture-associated IA population was formed by immigrants from the north who entered the region of contemporary Poland most likely at the beginning of the first millennium CE and mixed with autochthons. The presented results are in line with the hypothesis that assumes the genetic continuation between IA and MA periods in East-Central Europe.

NPM1 alternative transcripts are upregulated in acute myeloid and lymphoblastic leukemia and their expression level affects patient outcome.

BACKGROUND: Expression of the NPM1 gene, encoding nucleophosmin, is upregulated in cancers. Although more than ten NPM1 transcripts are known, the reports were usually limited to one predominant transcript. In leukemia, the NPM1 expression has not been widely studied so far. In acute myeloid leukemia (AML), the mutational status of the gene seems to play a pivotal role in carcinogenesis. Therefore, the aim of the study was to quantify alternative NPM1 transcripts in two types of acute leukemia, AML and ALL (acute lymphoblastic leukemia). METHODS: Using droplet digital PCR, we analyzed the levels of three protein-coding NPM1 transcripts in 66 samples collected from AML and ALL patients and 16 control samples. Using RNA-seq, we detected 8 additional NPM1 transcripts, including non-coding splice variants with retained introns. For data analysis, Welch two sample t-test, Pearson's correlation and Kaplan-Meier analysis were applied. RESULTS: The levels of the particular NPM1 transcripts were significantly different but highly correlated with each other in both leukemia and control samples. Transcript NPM1.1, encoding the longest protein (294 aa), had the highest level of accumulation and was one of the most abundant transcripts in the cell. Comparing to NPM1.1, the levels of the NPM1.2 and NPM1.3 transcripts, encoding a 265-aa and 259-aa proteins, were 30 and 3 times lower, respectively. All three NPM1 transcripts were proportionally upregulated in both types of leukemia compared to control samples. In AML, the levels of NPM1 transcripts decreased in complete remission and increased again with relapse of the disease. Low levels of NPM1.1 and NPM1.3 were associated with better prognosis. The contribution of non-coding transcripts to the total level of NPM1 gene seemed to be marginal, except for one short 5-end transcript accumulated at high levels in AML and control cells. Aberrant proportions of particular NPM1 splice variants could be linked to abnormal expression of genes encoding alternative splicing factors. CONCLUSIONS: The levels of the studied NPM1 transcripts were different but highly correlated with each other. Their upregulation in AML and ALL, decrease after therapy and association with patient outcome suggests the involvement of elevated NPM1 expression in the acute leukemia pathogenesis.

A mosaic genetic structure of the human population living in the South Baltic region during the Iron Age.

Despite the increase in our knowledge about the factors that shaped the genetic structure of the human population in Europe, the demographic processes that occurred during and after the Early Bronze Age (EBA) in Central-East Europe remain unclear. To fill the gap, we isolated and sequenced DNAs of 60 individuals from Kowalewko, a bi-ritual cemetery of the Iron Age (IA) Wielbark culture, located between the Oder and Vistula rivers (Kow-OVIA population). The collected data revealed high genetic diversity of Kow-OVIA, suggesting that it was not a small isolated population. Analyses of mtDNA haplogroup frequencies and genetic distances performed for Kow-OVIA and other ancient European populations showed that Kow-OVIA was most closely linked to the Jutland Iron Age (JIA) population. However, the relationship of both populations to the preceding Late Neolithic (LN) and EBA populations were different. We found that this phenomenon is most likely the consequence of the distinct genetic history observed for Kow-OVIA women and men. Females were related to the Early-Middle Neolithic farmers, whereas males were related to JIA and LN Bell Beakers. In general, our findings disclose the mechanisms that could underlie the formation of the local genetic substructures in the South Baltic region during the IA.

MLPA-Based Analysis of Copy Number Variation in Plant Populations.

Copy number variants (CNVs) are intraspecies duplications/deletions of large DNA segments (>1 kb). A growing number of reports highlight the functional and evolutionary impact of CNV in plants, increasing the need for appropriate tools that enable locus-specific CNV genotyping on a population scale. Multiplex ligation-dependent probe amplification (MLPA) is considered a gold standard in genotyping CNV in humans. Consequently, numerous commercial MLPA assays for CNV-related human diseases have been created. We routinely genotype complex multiallelic CNVs in human and plant genomes using the modified MLPA procedure based on fully synthesized oligonucleotide probes (90-200 nt), which greatly simplifies the design process and allows for the development of custom assays. Here, we present a step-by-step protocol for gene-specific MLPA probe design, multiplexed assay setup and data analysis in a copy number genotyping experiment in plants. As a case study, we present the results of a custom assay designed to genotype the copy number status of 12 protein coding genes in a population of 80 Arabidopsis accessions. The genes were pre-selected based on whole genome sequencing data and are localized in the genomic regions that display different levels of population-scale variation (non-variable, biallelic, or multiallelic, as well as CNVs overlapping whole genes or their fragments). The presented approach is suitable for population-scale validation of the CNV regions inferred from whole genome sequencing data analysis and for focused analysis of selected genes of interest. It can also be very easily adopted for any plant species, following optimization of the template amount and design of the appropriate control probes, according to the general guidelines presented in this paper.

Copy number variation of genes involved in the hepatitis C virus-human interactome.

Copy number variation (CNV) is a newly discovered form of intra-species genetic polymorphism that is defined as deletions or duplications of genome segments ranging from 1 kbp to several Mbp. CNV accounts for the majority of the genetic variation observed in humans (CNV regions cover more than 10% of the human genome); therefore, it may significantly influence both the phenotype and susceptibility to various diseases. Unfortunately, the impact of CNV on a number of diseases, including hepatitis C virus (HCV) infection, remains largely unexplored. Here, we analyzed 421 human genes encoding proteins that have been shown to interact with HCV proteins or genomic RNA (proteins from the HCV-human interactome). We found that 19 of the 421 candidate genes are located in putative CNV regions. For all of these genes, copy numbers were determined for European, Asiatic and African populations using the multiplex ligation-dependent amplification (MLPA) method. As a result, we identified 4 genes, IGLL1, MLLT4, PDPK1, PPP1R13L, for which the CN-genotype ranged from 1 to 6. All of these genes are involved in host-virus interaction; thus, their polymorphism has a potential impact on the development of HCV infection and/or therapy outcome.

Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification.

The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The presented method is simple, reliable and cost-effective. It can be easily introduced into clinical practice or developed to target both less-frequent mutations in the NPM1 gene and other cancer-related genes.

Copy number variation in the region harboring SOX9 gene in dogs with testicular/ovotesticular disorder of sex development (78,XX; SRY-negative).

Although the disorder of sex development in dogs with female karyotype (XX DSD) is quite common, its molecular basis is still unclear. Among mutations underlying XX DSD in mammals are duplication of a long sequence upstream of the SOX9 gene (RevSex) and duplication of the SOX9 gene (also observed in dogs). We performed a comparative analysis of 16 XX DSD and 30 control female dogs, using FISH and MLPA approaches. Our study was focused on a region harboring SOX9 and a region orthologous to the human RevSex (CanRevSex), which was located by in silico analysis downstream of SOX9. Two highly polymorphic copy number variable regions (CNVRs): CNVR1 upstream of SOX9 and CNVR2 encompassing CanRevSex were identified. Although none of the detected copy number variants were specific to either affected or control animals, we observed that the average number of copies in CNVR1 was higher in XX DSD. No copy variation of SOX9 was observed. Our extensive studies have excluded duplication of SOX9 as the common cause of XX DSD in analyzed samples. However, it remains possible that the causative mutation is hidden in highly polymorphic CNVR1.

An MLPA-based approach for high-resolution genotyping of disease-related multi-allelic CNVs.

Copy number variation has recently been recognized as an important type of genetic variation that modifies human phenotypes. Copy number variants (CNVs) are being increasingly associated with various human phenotypes and diseases. However, the lack of an appropriate method that allows fast, inexpensive and, most importantly, accurate CNVs genotyping significantly hampers CNV analysis. This limitation especially affects the analysis of multi-allelic CNVs that frequently modify various phenotypes. Recently, we developed a multiplex ligation-dependent probe amplification (MLPA)-based strategy for multiplex copy number genotyping and the validation of candidate CNV-miRNAs. Here we present the adaptation and optimization of this recently developed method for high-resolution genotyping of individual disease-related multi-allelic CNVs. We developed appropriate assays for three well-known and extensively studied CNVs: CNV-CCL3L1, CNV-DEFB, and CNV-UGT2B17, which have been associated with various human phenotypes including inflammation-related and infectious diseases. With the use of these assays we identified several general factors that allow to increase the resolution of the copy number genotyping. Performed experiments confirmed the high reproducibility and accuracy of the obtained genotyping results. The reliability of the results and relatively low per-genotype cost makes this strategy an attractive method for large-scale experiments such as genotype-phenotype association studies.

An MLPA-based strategy for discrete CNV genotyping: CNV-miRNAs as an example.

Copy number variation (CNV) has become well recognized in recent years. It has been estimated that common CNVs account for approximately 10% of the human genome and that they overlap hundreds of genes and other functional genetic elements. Although substantial progress in genome-wide CNV analysis has been made recently, there is still a need for a method that allows precise genotyping of selected CNVs. Here, we describe a novel strategy for CNV genotyping, taking advantage of the general principles of the multiplex ligation-dependent probe amplification (MLPA) method and short oligonucleotide probes, allowing easy custom design and generation of assays for almost any genomic region of interest. As a proof-of-concept, we developed two assays covering 17 candidate CNV regions that overlap human miRNA genes. Extensive quality control analysis demonstrated high reproducibility and reliability of the genotypes determined using our method. Detailed analysis of identified CNVs revealed that they are highly differentiated among the HapMap populations. The main advantages of the developed strategy include the simplicity of the assay design, its flexibility in terms of the selection of genomic regions, and its low cost (<$1-$10/genotype, depending on scale of experiment). These advantages make the presented strategy attractive for large-scale genetic analyses.