Exome sequencing from nanogram amounts of starting DNA: comparing three approaches

Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelectXT2 Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 µg of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input samples

Comparing Three Approaches

Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelectXT2 Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 µg of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input sample

Student’s class

The article presents the results of the implementation in the educational processadditional classes on medical subjects.В статье представлены результаты внедрения в образовательный процесс школьников 9–11-х классов внеклассных занятий на медицинскую тематику

Whole exome sequencing links dental tumor to an autosomal-dominant mutation in ANO5 gene associated with gnathodiaphyseal dysplasia and muscle dystrophies

Tumors of the jaws may represent different human disorders and frequently associate with pathologic bone fractures. In this report, we analyzed two affected siblings from a family of Russian origin, with a history of dental tumors of the jaws, in correspondence to original clinical diagnosis of cementoma consistent with gigantiform cementoma (GC, OMIM: 137575). Whole exome sequencing revealed the heterozygous missense mutation c.1067G \u3e A (p.Cys356Tyr) in ANO5 gene in these patients. To date, autosomal-dominant mutations have been described in the ANO5 gene for gnathodiaphyseal dysplasia (GDD, OMIM: 166260), and multiple recessive mutations have been described in the gene for muscle dystrophies (OMIM: 613319, 611307); the same amino acid (Cys) at the position 356 is mutated in GDD. These genetic data and similar clinical phenotypes demonstrate that the GC and GDD likely represent the same type of bone pathology. Our data illustrate the significance of mutations in single amino-acid position for particular bone tissue pathology. Modifying role of genetic variations in another gene on the severity of the monogenic trait pathology is also suggested. Finally, we propose the model explaining the tissue-specific manifestation of clinically distant bone and muscle diseases linked to mutations in one gene

Cost-effectiveness analysis of surgical treatment of patients with early-stage breast cancer luminal subtype A in order to optimize the treatment of organic-savings

Evaluate the cost effectiveness of different options of surgical treatment in patients with early breast cancer luminal subtype A. An analysis of 200 patients with T1N0M0 stage breast cancer with luminal subtype A. The patients were divided into 4 groups: 1 gr. - mastectomy- 50,2 gr. - resection of breast cancer - 50,3 gr. - sectoral resection with lymphadenectomy stadiruyuschey - 50,4 gr. - sectoral resection with sentinel lymph node biopsy. Follow-up was 5 years. As a result, it was found that survival and distant metastasis in patients with breast cancer groups stage T1N0M0 after mastectomy and sparing surgery is no different.Цель работы -оценить экономическую эффективность различных вариантов хирургического лечения у больных с ранним РМЖ люминальным подтипом А. Проведен анализ 200 пациенток с T1N0M0 стадией РМЖ с люминальным типом А. Пациентки разделены на 4 группы: 1 гр. - мастэктомия - 50; 2 гр. - радикальная резекция молочной железы - 50,3 гр. - секторальная резекция со стадирующей лимфодиссекцией - 50,4 гр. - секторальная резекция с биопсией сторожевого лимфоузла. Сроки наблюдения составили 5 лет. В результате нами установлено, что выполнение секторальной резекции с биопсией сигнального лимфоузла у пациенток с раком молочной железы T1N0M0 стадией (люминальный подтипом А) позволяет сократить длительность стационарного лечения и соответственно уменьшить стоимость стационарного лечения, при сопоставимой общей и безрецидивной выживаемости

Von Tn5-basierter DNS-Fragmentierungseffizienz und Beurteilung des 'tagmentation site indexing'-Ansatzes für die kontiguität-bewahrende Sequenzierung

ACKNOWLEDGMENTS 6 ABBREVIATIONS 7 SUMMARY 8 Zusammenfassung 9 INTRODUCTION 10 Sequencing gaps: loss of contiguity information 10 Solutions for contiguity preserving sequencing 13 Extending the read length: single molecule sequencing 13 Experimental approaches to link short reads 16 Subselection of a particular part of a genome 16 Random subselection of a part of a genome 20 Cloning pools 20 Fragment pools 22 CPT-seq 24 Proximity ligation strategies 27 Paired indexing of fragmentation sites 29 MATERIALS AND METHODS 34 Materials 34 Chemicals 34 Hardware and Plastic 36 Enzymes 38 Kits 39 Oligonucleotides 39 Plasmids 43 Solutions 43 Methods 45 Tn5 Production 45 Bacterial Stab 45 Glycerol Stocks 45 Plasmid DNA Preparation 45 Sequencing 46 Protein expression 46 Transformation T7 Express lysY/Iq Competent E. coli cells 46 Overexpression 46 Tn5 purification 47 Preparation of Chitin Magnetic Beads 47 Binding 47 Cleavage 48 Elution 48 Protein concentration and buffer exchange 49 Transposase activity assay 49 Fragmentation Efficiency Assay (FEA) 50 Transposon 50 Transposome assembly 50 Tagmentation template 51 Tagmentation 51 qPCR 52 Visual fragments size analysis 52 PITS Library Preparation 53 Transposome assembly 53 Phage Lambda genomic DNA 53 Tagmentation 53 Dilution 54 Oligo replacement and gap-filling reaction 54 Amplification 55 Flow cell loading and sequencing 56 Sequencing analysis 56 Preparation of Alternative Transposon structures 57 Synthetic Lampion Transposon 57 Preparation of PCR Products of Different Lengths 57 Transposon Insertion with Epicentre Transposase 58 Transposon Insertion with In-House Tn5 Transposase 59 RESULTS AND DISCUSSION 60 Preface 60 Tagmentation sites indexing approach for contiguity-preserving sequencing 60 NGS library preparation from amol amounts of DNA and non-residual loading on Illumina sequencing flowcell 61 Tagmentation 63 Dilution of the tagmentation reaction 64 Gap repair reaction 65 Amplification and loading on a flowcell 67 Recommended experimental setup 68 Proof-of principle paired indexing experiment 71 Indexing scheme 71 Template selection 73 PITS libraries sequencing 73 Preparation of in-house Tn5 Transposase 78 Tn5 Transposase expression and purification 78 Transposase activity assay 79 Fragmentation efficiency assay (FEA) 79 Discussion of the PITS protocol 86 REFERENCES 89 SUPPLEMENTARY 100A novel contiguity-preserving sequencing approach was developed and tested – paired indexing at tagmentation sites (PITS). The idea of the method is to use paired indices to individually label pairs of DNA molecule ends originating at tagmentation sites. Indices from the same pair on the ends of two sequencing library fragments would mean that those fragments were next to each other in the original molecule. Thus after getting separated in the way it occurs in a standard sequencing library preparation protocol library, fragments after sequencing would be assembled again and the contiguity of the sequence would be restored. Convenient system for testing of the PITS method was chosen and proof-of-principle experiment was performed. Though few, scaffolds containing up to 4 subsequent library molecules were assembled. Some of the constraints of the PITS approach were revealed and optimization possibilities for future work were determined. A patent application describing the PITS protocol is in preparation. During the course of this PhD, an efficient method for preparation of sequencing library from amol amounts of tagmentation products has been established – post tagmentation ultra low input (PTULI) protocol. The method aims at preserving possibly all tagmentation fragments throughout sequencing library preparation process. The developed protocol provides detailed instructions on the controls setup and guidelines for subsequent non- residual loading of the PTULI library on a sequencer. The PTULI library preparation strategy is suitable for PITS, and is also of value for other minute input material sequencing applications. To make contiguity-preserving sequencing work possible, in-house Tn5 transposase was prepared. Applications of this enzyme within the settings other than those in commercially available kits are hardly known and further development of the PITS approach to a great extent depends on the potential of this enzyme. That is why in parallel to the PITS method itself, Tn5 properties were studied. An electrophoresis free assay for characterizing Tn5 transposomes fragmentation efficiency was developed and published [Rykalina et al., 2017]. This assay is a convenient monitoring system for the setup of tagmentation protocols and for optimization experiments.Es wurde ein neuartiger Kontiguität-bewahrender Sequenzierungsansatz entwickelt und getestet: gepaarte Indizierung an Tagmentationsstellen (Paired Indexing at Tagmentation Sites - PITS). Die Idee der Methode besteht darin, die Paare der DNA-Molekül-Enden, die ursprünglich von Tagmentationsstellen stammen, individuell mit gepaarten Indizes zu markieren. Gleiche Indizes an den Enden der zwei sequenzierten Bibliotheksfragmente würde bedeuten, dass diese Fragmente im ursprünglichen Molekül nebeneinander angeordnet waren. Dank dieser Methode werden die Fragmente, die während der Herstellung von Sequenzierungsbibliotheken getrennt wurden, wieder zusammengesetzt und damit die Kontiguität der Sequenz der ursprünglichen DNA-Moleküle wiederhergestellt. Es wurde ein praktisches System für die Prüfung der PITS-Methode gewählt und ein proof-of-principle Experiment durchgeführt. DNA Ketten mit bis zu vier nachfolgenden Bibliotheksmolekülen wurden zusammengebaut. Einige der Einschränkungen des PITS-Ansatzes wurden aufgedeckt und Optimierungsmöglichkeiten für zukünftige Arbeiten ermittelt. Eine Patentanmeldung, die das PITS-Protokoll beschreibt, ist in Vorbereitung. Im Rahmen der Arbeit wurde ein effizientes Verfahren zur Herstellung von Sequenzierungsbibliotheken aus amol-Mengen von Tagmentationsprodukten etabliert, das Post-Tagmentation Ultra Low Input (PTULI) Protokoll. Die Methode zielt darauf ab, möglichst alle Tagmentierungsfragmente während des gesamten Prozesses zur Vorbereitung der Sequenzierungsbibliotheken zu bewahren. Das entwickelte Protokoll enthält detaillierte Anweisungen zum Steuerungs-Setup und Richtlinien für die anschließende komplette Beladung der PTULI-Bibliothek auf einem Sequenzer. Die PTULI- Bibliotheksvorbereitungsstrategie eignet sich nicht nur für PITS, sondern auch für andere Sequenzierungsanwendungen mit geringen Input. Um dieses Projekt zu ermöglichen, wurde eine eigene Tn5-Transposase hergestellt. Anwendungen dieses Enzyms in anderen settings als in handelsüblichen Kits sind kaum bekannt und die Weiterentwicklung des PITS-Ansatzes hängt weitgehend von dem Potenzial dieses Enzyms ab. Deshalb wurden parallel zur PITS-Methode selbst Tn5-Eigenschaften untersucht. Ein elektrophoresefreier Assay zur Charakterisierung von Tn5-Transposomen Fragmentierungseffizienz wurde entwickelt und veröffentlicht [Rykalina et al., 2017]. Dieser Assay bietet ein bequemes Monitoring-System für den Aufbau von Tagmentationsprotokollen und für Optimierungsexperimente

The Low Carbon Trend from a Sustainability Perspective: Limiting Greenhouse Gas Emissions

Climate change is the main global risk in the next 10 years, and international organizations, businesses and governments are paying more and more attention to decarbonization strategies and energy transition. In line with the general trend, the popularity of the climate agenda in Russia has grown rapidly in recent years. Until recently, many Russian companies calculated their carbon footprint, planned the development and implementation of climate strategies, and the state was actively developing the regulatory infrastructure for low-carbon development. Banking organizations have also announced climate goals and implemented decarbonization activities. However, a sharp change in the geopolitical and economic situation calls into question the future of the national climate agenda. At first glance, it may seem that the importance of low-carbon development principles has decreased, and banks, which have just begun their journey within the framework of the sustainable development agenda, have lost momentum. Increasingly, there are opinions that the main driver for the development of climate strategies, the request of international investors, has lost its relevance

Conceptual approaches to improvement of the sustainability management mechanism of the agrarian sphere development of the region in the Russian Federation

In the framework of the study, the authors consider conceptual approaches to improve the management system of agrarian sphere development of regions in the realities of this industry functioning in the Russian Federation. Agrarian sphere is shown in aspects of socio-economic territorial system with its subsystems. The transition mechanisms of the agrarian sphere to the rails of sustainable development due to the choice of priority development directions within the life cycle are presented. The necessity of applying an integrated approach to the solution of industrial and social problems of the agricultural sector of the country is proved and reasoned

Issues of agriculture digitalization in the Russian Federation (legal aspect)

The President of the Russian Federation has set the task of achieving “digital maturity” of the key economic sectors, including agriculture, by 2030. At the same time, the digitalization of the agro-industrial complex is clearly lagging behind the pace of digital transformation in other sectors of the national economy. There are several reasons for this: the regulatory standards of the agricultural crops cultivation and the farm animals breeding are not perfect; the legal regulation of digitalization in the field of both production and agriculture is fragmented; the use of digital tools and information technologies in agricultural activities appears to be quite complicated. The article not only analyzes modern acts affecting the digital transformation of agriculture in the Russian Federation (including acts of the Ministry of Agriculture of the Russian Federation) and the problems of their application, but also suggests measures aimed to stimulate agricultural producers and food-processing companies, introducing innovative digital technologies, as well as provides recommendations for the use of special legal regimes that entered into force in 2021

State regulation of supply chains through the introduction of goods marking

Among the most acute problems of the modern economy is not only reducing the cost and improving the efficiency of supply chains, but also their protection from the influence of the shadow economy. In this case, the greatest threat is the illicit trafficking of industrial products. Authors analyze state regulation of supply chains in Russia and abroad, consider the prospects for the development of digital marking and traceability of goods as a method of state regulation of supply chains. The article emphasizes that the existing marking system contains a number of shortcomings. The authors propose mechanisms to improve state regulation of supply chains in modern conditions