RESUMEN
Metagenomics is a powerful tool to study marine microbial communities. However, obtaining high-quality environmental DNA suitable for downstream sequencing applications is a challenging task. The quality and quantity of isolated DNA heavily depend on the choice of purification procedure and the type of sample. Selection of an appropriate DNA isolation method for a new type of material often entails a lengthy trial and error process. Further, each DNA purification approach introduces biases and thus affects the composition of the studied community. To account for these problems and biases, we systematically investigated efficiency of DNA purification from three types of samples (water, sea sediment, and digestive tract of a model invertebrate Magallana gigas) with eight commercially available DNA isolation kits. For each kit-sample combination we measured the quantity of purified DNA, extent of DNA fragmentation, the presence of PCR-inhibiting contaminants, admixture of eukaryotic DNA, alpha-diversity, and reproducibility of the resulting community composition based on 16S rRNA amplicons sequencing. Additionally, we determined a "kitome", e.g., a set of contaminating taxa inherent for each type of purification kit used. The resulting matrix of evaluated parameters allows one to select the best DNA purification procedure for a given type of sample.
Asunto(s)
Benchmarking , ADN , ARN Ribosómico 16S/genética , Reproducibilidad de los Resultados , ADN/genética , Análisis de Secuencia de ADN/métodos , Metagenómica/métodos , ADN Bacteriano/genéticaRESUMEN
IgA nephropathy (IgAN) is an autoimmune disorder which is believed to be non-monogenic. We performed an exome-wide association study of 70 children with IgAN and 637 healthy donors. The HLA allele frequencies were compared between the patients and healthy donors from the bone marrow registry of the Pirogov University. We tested 78,020 gene markers for association and performed functional enrichment analysis and transcription factor binding preference detection. We identified 333 genetic variants, employing three inheritance models. The most significant association with the disorder was observed for rs143409664 (PRAG1) in the case of the additive and dominant models (PBONF = 1.808 × 10-15 and PBONF = 1.654 × 10-15, respectively), and for rs13028230 (UBR3) in the case of the recessive model (PBONF = 1.545 × 10-9). Enrichment analysis indicated the strongly overrepresented "immune system" and "kidney development" terms. The HLA-DQA1*01:01:01G allele (p = 0.0076; OR, 2.021 [95% CI, 1.322-3.048]) was significantly the most frequent among IgAN patients. Here, we characterized, for the first time, the genetic background of Russian IgAN patients, identifying the risk alleles typical of the population. The most important signals were detected in previously undescribed loci.
Asunto(s)
Glomerulonefritis por IGA , Ubiquitina-Proteína Ligasas , Niño , Humanos , Estudios de Casos y Controles , Exoma/genética , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Glomerulonefritis por IGA/genética , Glomerulonefritis por IGA/diagnóstico , Polimorfismo de Nucleótido Simple , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
This article is devoted to a brief review of the modelling of liquid membrane separation methods, such as emulsion, supported liquid membranes, film pertraction, and three-phase and multi-phase extraction. Mathematical models and comparative analyses of liquid membrane separations with different flow modes of contacting liquid phases are presented. A comparison of the processes of conventional and liquid membrane separations is carried out under the following assumptions: mass transfer is described by the traditional mass transfer equation; the equilibrium distribution coefficients of a component passing from one of the phases to another are constant. It is shown that, from the point of view of mass transfer driving forces, emulsion and film pertraction liquid membrane methods have advantages over the conventional conjugated extraction stripping method, when the mass-transfer efficiency of the extraction stage is significantly higher than the efficiency of the stripping stage. The comparison of the supported liquid membrane with conjugated extraction stripping showed that when mass-transfer rates on the extraction and stripping sides are different, the liquid membrane method is more efficient, while when they are equal to each other, both processes demonstrate the same results. The advantages and disadvantages of liquid membrane methods are discussed. The main disadvantages of liquid membrane methods-low throughput and complexity-can be overcome by using modified solvent extraction equipment to carry out liquid membrane separations.
RESUMEN
Pseudo-liquid membranes are extraction devices in which a liquid membrane phase is retained in an apparatus consisting of two interconnected chambers while feed and stripping phases pass through the stationary liquid membrane phase as mobile phases. The organic phase of the liquid membrane sequentially contacts the aqueous phases of the feed and stripping solutions in the extraction and stripping chambers, recirculating between them. This extraction separation method, called multiphase pseudo-liquid membrane extraction, can be implemented using traditional extraction equipment: extraction columns and mixer-settlers. In the first case, the three-phase extraction apparatus consists of two extraction columns connected at the top and bottom by recirculation tubes. In the second case, the three-phase apparatus consists of a recycling close-loop, which includes two mixer-settler extractors. In this study, the extraction of copper from sulfuric acid solutions in two-column three-phase extractors was experimentally studied. A 20% solution of LIX-84 in dodecane was used as the membrane phase in the experiments. It was shown that the extraction of copper from sulfuric acid solutions in the apparatuses studied was controlled by the interfacial area in the extraction chamber. The possibility of the purification of sulfuric acid wastewaters from copper using three-phase extractors is shown. To increase the degree of extraction of metal ions, it is proposed to equip two-column three-phase extractors with perforated vibrating discs. To further increase the efficiency of extraction using the pseudo-liquid membrane method, it is proposed to use multistage processes. The mathematical description of multistage three-phase pseudo-liquid membrane extraction is discussed.
RESUMEN
To promote the implementation of liquid membrane separations in industry, we have previously proposed extraction methods called three- and multi-phase extraction. The three-phase multi-stage extraction is carried out in a cascade of bulk liquid membrane separation stages, each comprising two interconnected (extraction and stripping) chambers. The organic liquid membrane phase recycles between the chambers within the same stage. In multi-phase extraction, each separation stage includes a scrubbing chamber, located between the extraction and stripping chambers. The three- and multi-phase multi-stage extraction technique can be realized either in a series of mixer-settler extractors or in special two- or multi-chamber extraction apparatuses, in which the convective circulation of continuous membrane phase between the chambers takes place due to the difference in emulsion density in the chambers. The results of an experimental study of the extraction of phenol from sulfuric acid solutions in the three-phase extractors with convective circulation of continuous membrane phase are presented. Butyl acetate was used as an extractant. The stripping of phenol from the organic phase was carried out with 5-12% NaOH aqueous solutions. The prospects of using three-phase extractors for wastewater treatment from phenol are shown. An increase in the efficiency of three-phase extraction can be achieved by carrying out the process in a cascade of three-phase apparatuses.
RESUMEN
Exome sequencing is becoming a routine in health care, because it increases the chance of pinpointing the genetic cause of an individual patient's condition and thus making an accurate diagnosis. It is important for facilities providing genetic services to keep track of changes in the technology of exome capture in order to maximize throughput while reducing cost per sample. In this study, we focused on comparing the newly released exome probe set Agilent SureSelect Human All Exon v8 and the previous probe set v7. In preparation for higher throughput of exome sequencing using the DNBSEQ-G400, we evaluated target design, coverage statistics, and variants across these two different exome capture products. Although the target size of the v8 design has not changed much compared to the v7 design (35.24 Mb vs 35.8 Mb), the v8 probe design allows you to call more of SNVs (+ 3.06%) and indels (+ 8.49%) with the same number of raw reads per sample on the common target regions (34.84 Mb). Our results suggest that the new Agilent v8 probe set for exome sequencing yields better data quality than the current Agilent v7 set.
Asunto(s)
Exoma , Secuenciación de Nucleótidos de Alto Rendimiento , Exones , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación INDEL , Secuenciación del ExomaRESUMEN
We report incidence and deep molecular characteristics of lineage switch in 182 pediatric patients affected by B-cell precursor acute lymphoblastic leukemia (BCP-ALL), who were treated with blinatumomab. We documented six cases of lineage switch that occurred after or during blinatumomab exposure. Therefore, lineage conversion was found in 17.4% of all resistance cases (4/27) and 3.2% of relapses (2/63). Half of patients switched completely from BCP-ALL to CD19-negative acute myeloid leukemia, others retained CD19-positive B-blasts and acquired an additional CD19-negative blast population: myeloid or unclassifiable. Five patients had KMT2A gene rearrangements; one had TCF3::ZNF384 translocation. The presented cases showed consistency of gene rearrangements and fusion transcripts across initially diagnosed leukemia and lineage switch. In two of six patients, the clonal architecture assessed by IG/TR gene rearrangements was stable, while in others, loss of clones or gain of new clones was noted. KMT2A-r patients demonstrated very few additional mutations, while in the TCF3::ZNF384 case, lineage switch was accompanied by a large set of additional mutations. The immunophenotype of an existing leukemia sometimes changes via different mechanisms and with different additional molecular changes. Careful investigation of all BM compartments together with all molecular -minimal residual disease studies can lead to reliable identification of lineage switch.
Asunto(s)
Anticuerpos Biespecíficos , Leucemia de Células B , Leucemia Linfocítica Crónica de Células B , Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/uso terapéutico , Niño , Humanos , Leucemia de Células B/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Mieloide Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Factores de Transcripción/genética , Translocación GenéticaRESUMEN
Three novel strains of Gram-stain-negative, obligately anaerobic, spore-forming straight or slightly curved rods with pointed ends occurring singly or in pairs were isolated from the faeces of healthy human children. The strains were characterized by mesophilic fermentative metabolism and production of acetate, ethanol and H2 as the end metabolic products. Strains ASD3451 and ASD5720T were motile, fermented lactose and raffinose, and weakly fermented maltose. Strain ASD4241T was non-motile and did not ferment the carbohydrates listed above but fermented starch. Strains ASD3451 and ASD5720T shared average nucleotide identity higher than 98.5â% with each other, while ASD4241T had only 88.5-89â% identity to them. Based on phylogenetic and chemotaxonomic analyses, we propose Diplocloster agilis gen. nov., sp. nov. (ASD5720T=JCM 34353T=VKM B-3497T) and Diplocloster modestus sp. nov. (ASD4241T=JCM 34351T=VKM B-3498T) within the family Lachnospiraceae.
Asunto(s)
Heces/microbiología , Firmicutes/clasificación , Filogenia , Anaerobiosis , Técnicas de Tipificación Bacteriana , Composición de Base , Niño , ADN Bacteriano/genética , Ácidos Grasos/química , Firmicutes/aislamiento & purificación , Humanos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Human exome sequencing is a classical method used in most medical genetic applications. The leaders in the field are the manufacturers of enrichment kits based on hybridization of cRNA or cDNA biotinylated probes specific for a genomic region of interest. Recently, the platforms manufactured by the Chinese company MGI Tech have become widespread in Europe and Asia. The reliability and quality of the obtained data are already beyond any doubt. However, only a few kits compatible with these sequencers can be used for such specific tasks as exome sequencing. We developed our own solution for library pre-capture pooling and exome enrichment with Agilent probes. In this work, using a set of the standard benchmark samples from the Platinum Genome collection, we demonstrate that the qualitative and quantitative parameters of our protocol which we called "RSMU_exome" exceed those of the MGI Tech kit. Our protocol allows for identifying more SNV and indels, generates fewer PCR duplicates, enables pooling of more samples in a single enrichment procedure, and requires less raw data to obtain results comparable with the MGI Tech's protocol. The cost of our protocol is also lower than that of MGI Tech's solution.
Asunto(s)
Sondas de ADN , Secuenciación del Exoma/normas , Composición de Base , Humanos , Mutación INDEL , Polimorfismo de Nucleótido Simple , Secuenciación del Exoma/economíaRESUMEN
Background. This study deals with a rare (orphan) monogenic connective tissue disorder - Ehlers-Danlos syndrome kyphoscoliotic type 2 (EDSKS2). Kyphoscoliotic type 2 Ehlers-Danlos syndrome is an autosomal recessive disorder caused by mutations in the FKBP14 gene (7p14.3), which encodes the FKBP22 protein. According to the 2017 classification, this type is in group seven - collagen spatial structure and cross-linking defects. We present results of clinical examination and molecular genetic analysis for five patients with age varying from two to fifteen years. Methods. Five patients were examined using clinical and laboratory methods. DNA samples used for the analysis were extracted from whole blood samples using a Wizard® Genomic DNA Purification Kit (Promega, USA) according to the manufacturer's protocol. Results. The major clinical findings were kyphoscoliosis, early motor development delay, muscular weakness, hypotonia and hearing loss. Molecular genetic analysis detected a homozygous c.362dupC duplication in exon 3 of the FKBP14 gene in all five patients. This mutation is common in various countries. Differential diagnostics were carried out to exclude other Ehlers-Danlos syndrome types and myopathies. Conclusions. Literature analysis and examination of five EDSKS2 patients demonstrated the involvement of major organs and systems, such as joints, spine, muscles, cardiovascular system, respiratory system, hearing, and vision, into the pathological process. Kidney mobility increases and nephroptosis seems to be secondary caused by muscular weakness. During molecular genetic analysis, to verify EDSKS2 it is recommended to initially search for the c.362dupC duplication, which appears to be common in European countries, including Russia.
Asunto(s)
Síndrome de Ehlers-Danlos , Adolescente , Niño , Preescolar , Síndrome de Ehlers-Danlos/diagnóstico , Síndrome de Ehlers-Danlos/genética , Exones , Humanos , Mutación , Isomerasa de Peptidilprolil/genética , FenotipoRESUMEN
Here we present the devised BC-store-a program for analyzing and selecting sets of barcodes for sequencing on platforms manufactured by MGI Tech (China). The app is available as an open source in Python3 and as a desktop version. The application allows analyzing the compatibility of barcodes on a single lane of a flow cell in a set in the case of equal and arbitrary fractions. In addition, with the help of this tool barcodes can be added to an existing set with custom share options. In this paper we describe how BC-store works for different tasks and consider the effectiveness of using BC-store in sequence lab routine tasks.
Asunto(s)
Código de Barras del ADN Taxonómico/instrumentación , Código de Barras del ADN Taxonómico/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Programas Informáticos , Algoritmos , Secuencia de Bases/genética , HumanosRESUMEN
Countercurrent liquid-liquid chromatographic techniques (CCC), similar to solvent extraction, are based on the different distribution of compounds between two immiscible liquids and have been most widely used in natural product separations. Due to its high load capacity, low solvent consumption, the diversity of separation methods, and easy scale-up, CCC provides an attractive tool to obtain pure compounds in the analytical, preparative, and industrial-scale separations. This review focuses on the steady-state and non-steady-state CCC separations ranging from conventional CCC to more novel methods such as different modifications of dual mode, closed-loop recycling, and closed-loop recycling dual modes. The design and modeling of various embodiments of CCC separation processes have been described.
Asunto(s)
Productos Biológicos/análisis , Distribución en Contracorriente , Solventes/químicaRESUMEN
Industrial separation technologies can be improved and greatly simplified by using the methods of counter-current chromatography (CCC). We have previously proposed the use of currently available solvent extraction equipment (a series of multistage columns, a cascade of centrifugal mixer-settler extractors) as large-scale CCC devices. For industrial separations, the application of closed-loop recycling counter-current chromatography (CLR CCC) methods seems to be the most promising. To improve the performance of the CLR CCC separations, semi-continuous three-stage processes (1 - continuous loading of the mixture solution over a definite time; 2 - separation of solutes in recycling closed-loop; 3 - elution of the fractions of the separated solutes with the mobile phase) can be used. The purpose of this study is to present a simple and easy to use mathematical model allowing the simulation and design of various options for implementing such separation processes and analyze the influence of its main parameters on separation efficiency.
Asunto(s)
Técnicas de Química Analítica/métodos , Distribución en Contracorriente , Industrias/métodos , Modelos Teóricos , Técnicas de Química Analítica/instrumentación , Industrias/instrumentación , Soluciones/químicaRESUMEN
Non-invasive prenatal testing (NIPT) for aneuploidy on Chromosomes 21 (T21), 18 (T18) and 13 (T13) is actively used in clinical practice around the world. One of the limitations of the wider implementation of this test is the high cost of the analysis itself, as high-throughput sequencing is still relatively expensive. At the same time, there is an increasing trend in the length of reads yielded by sequencers. Since extracellular DNA is short, in the order of 140-160 bp, it is not possible to effectively use long reads. The authors used high-performance sequencing of cell-free DNA (cfDNA) libraries that went through additional stages of enzymatic fragmentation and random ligation of the resulting products to create long chimeric reads. The authors used a controlled set of samples to analyze a set of cfDNA samples from pregnant women with a high risk of fetus aneuploidy according to the results of the first trimester screening and confirmed by invasive karyotyping of the fetus using laboratory and analytical approaches developed by the authors. They evaluated the sensitivity, specificity, PPV (positive predictive value), and NPV (negative predictive value) of the results. The authors developed a technique for constructing long chimeric reads from short cfDNA fragments and validated the test using a control set of extracellular DNA samples obtained from pregnant women. The obtained sensitivity and specificity parameters of the NIPT developed by the authors corresponded to the approaches proposed earlier (99.93% and 99.14% for T21; 100% and 98.34% for T18; 100% and 99.17% for T13, respectively).
Asunto(s)
Aneuploidia , Ácidos Nucleicos Libres de Células/sangre , Síndrome de la Trisomía 13/genética , Síndrome de la Trisomía 18/genética , Adulto , Ácidos Nucleicos Libres de Células/genética , Quimera/genética , Cromosomas Humanos Par 13/genética , Cromosomas Humanos Par 18/genética , Cromosomas Humanos Par 21/genética , Femenino , Humanos , Embarazo , Diagnóstico Prenatal , Síndrome de la Trisomía 13/sangre , Síndrome de la Trisomía 13/patología , Síndrome de la Trisomía 18/sangre , Síndrome de la Trisomía 18/patologíaRESUMEN
The MGISEQ-2000 developed by MGI Tech Co. Ltd. (a subsidiary of the BGI Group) is a new competitor of such next-generation sequencing platforms as NovaSeq and HiSeq (Illumina). Its sequencing principle is based on the DNB and the cPAS technologies, which were also used in the previous version of the BGISEQ-500 device. However, the reagents for MGISEQ-2000 have been refined and the platform utilizes updated software. The cPAS method is an advanced technology based on the cPAL previously created by Complete Genomics. In this paper, the authors compare the results of the whole-genome sequencing of a DNA sample from a Russian female donor performed on MGISEQ-2000 and Illumina HiSeq 2500 (both PE150). Two platforms were compared in terms of sequencing quality, number of errors and performance. Additionally, we performed variant calling using four different software packages: Samtools mpileaup, Strelka2, Sentieon, and GATK. The accuracy of SNP detection was similar in the data generated by MGISEQ-2000 and HiSeq 2500, which was used as a reference. At the same time, a separate indel analysis of the overall error rate revealed similar FPR values and lower sensitivity. It may be concluded with confidence that the data generated by the analyzed sequencing systems is characterized by comparable magnitudes of error and that MGISEQ-2000 and HiSeq 2500 can be used interchangeably for similar tasks like whole genome sequencing.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Secuenciación Completa del Genoma , Bases de Datos Genéticas , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Polimorfismo de Nucleótido Simple/genética , Control de Calidad , Secuenciación Completa del Genoma/normasRESUMEN
Closed-loop recycling dual-mode counter-current chromatography (CLR DM CCC) processes consist of two successive separation stages: separation of solutes in the recycling closed-loop with mobile x-phase and separation of solutes in the counter-current mode with mobile y-phase. Several variants of the implementation of this separation method can be developed: the closed-loop recycling stage may consist of one or several successive separation steps; all components of a mixture can pass through both stages of separation or individual components may be withdrawn from the system at different stages. In this study, such separation processes are theoretically investigated, and simple equations for simulation presented. These equations can help to simulate and select a suitable separation scheme for a given mixture of components. Several examples of separation by CLR DM CCC method are discussed. Examples of simulation are presented in "Mathcad" program. It is shown that CLR DM CCC allows separation of solutes with similar partition coefficients and separation of complex mixtures containing solutes with widely different partition coefficients, providing concentrated fractions of the separated solutes.
Asunto(s)
Técnicas de Química Analítica/instrumentación , Técnicas de Química Analítica/métodos , Distribución en Contracorriente/instrumentación , Mezclas Complejas , Simulación por Computador , Modelos Teóricos , Reciclaje , Soluciones/químicaRESUMEN
Closed-loop recycling countercurrent chromatography (CLR CCC) with multiple sample injection has been shown to provide simultaneous concentration and separation of target compounds from multicomponent mixtures. Previous analysis of CLR CCC with multiple sample injections has been limited to the ideal recycling model, which neglects the effects caused by the pump and connecting lines. In this study, an analysis of the process is carried out based on the non-ideal recycling model: recycling chromatograms at two points of the closed-loop - the inlet of the column (A) and the outlet of the column (B) - are considered. The sample is repeatedly introduced at the inlet of the column when the circulating peak of target compound passes point A. Analytical expressions are developed, allowing the design and simulation of different variants of simultaneous separation and concentration of target compounds from multicomponent mixtures. Examples of separation of target compounds from three and five-component mixtures are discussed. Experimental results are presented demonstrating a reasonable agreement between the theory and the experiment. Due to its ability to concentrate individual solutes, CRL CCC with multiple sample injections can become an efficient analytical method to determine minor components in complex mixtures.
Asunto(s)
Aspirina/aislamiento & purificación , Cafeína/aislamiento & purificación , Mezclas Complejas/química , Distribución en Contracorriente/instrumentación , Distribución en Contracorriente/métodos , ReciclajeRESUMEN
Two well known approaches are considered to analyze the processes of counter-current and dual counter-current chromatography: the longitudinal mixing cell model and the Craig's counter-current distribution model. The cell model represents perfectly mixed, equally sized cells in series. The number of cells characterizes the rates of longitudinal mixing in the stationary and mobile phases. In the eluting counter-current distribution (CCD) model, the CCC process is considered as a continuous form of Craig's counter-current distribution. For a cascade of equilibrium stages theoretical elution profiles of the CCC process by using the CCD and cell model approaches have been compared. It is shown that in general, distribution functions of the CCD and cell models differ. It is established that the distribution of a solute between two solvent phases in the dual CCC process is determined by the extraction factor c, the total number of equilibrium stages n and the position of the sample inlet m by the equation Q(x)=(1-c(m))/(1-c(n+1)) with c=F(2)K(D)/F(1) (K(D), F(1), F(2) and Q(x) are the distribution constant, the phase flow-rates and the portion of solute eluted by the first phase, respectively).