Taxonomic classification of DNA sequences beyond sequence similarity using deep neural networks.

Taxonomic classification, that is, the assignment to biological clades with shared ancestry, is a common task in genetics, mainly based on a genome similarity search of large genome databases. The classification quality depends heavily on the database, since representative relatives must be present. Many genomic sequences cannot be classified at all or only with a high misclassification rate. Here we present BERTax, a deep neural network program based on natural language processing to precisely classify the superkingdom and phylum of DNA sequences taxonomically without the need for a known representative relative from a database. We show BERTax to be at least on par with the state-of-the-art approaches when taxonomically similar species are part of the training data. For novel organisms, however, BERTax clearly outperforms any existing approach. Finally, we show that BERTax can also be combined with database approaches to further increase the prediction quality in almost all cases. Since BERTax is not based on similar entries in databases, it allows precise taxonomic classification of a broader range of genomic sequences, thus increasing the overall information gain.

Robust direct digital-to-biological data storage in living cells.

DNA has been the predominant information storage medium for biology and holds great promise as a next-generation high-density data medium in the digital era. Currently, the vast majority of DNA-based data storage approaches rely on in vitro DNA synthesis. As such, there are limited methods to encode digital data into the chromosomes of living cells in a single step. Here, we describe a new electrogenetic framework for direct storage of digital data in living cells. Using an engineered redox-responsive CRISPR adaptation system, we encoded binary data in 3-bit units into CRISPR arrays of bacterial cells by electrical stimulation. We demonstrate multiplex data encoding into barcoded cell populations to yield meaningful information storage and capacity up to 72 bits, which can be maintained over many generations in natural open environments. This work establishes a direct digital-to-biological data storage framework and advances our capacity for information exchange between silicon- and carbon-based entities.

A unified dinucleotide alphabet describing both RNA and DNA structures.

By analyzing almost 120 000 dinucleotides in over 2000 nonredundant nucleic acid crystal structures, we define 96+1 diNucleotide Conformers, NtCs, which describe the geometry of RNA and DNA dinucleotides. NtC classes are grouped into 15 codes of the structural alphabet CANA (Conformational Alphabet of Nucleic Acids) to simplify symbolic annotation of the prominent structural features of NAs and their intuitive graphical display. The search for nontrivial patterns of NtCs resulted in the identification of several types of RNA loops, some of them observed for the first time. Over 30% of the nearly six million dinucleotides in the PDB cannot be assigned to any NtC class but we demonstrate that up to a half of them can be re-refined with the help of proper refinement targets. A statistical analysis of the preferences of NtCs and CANA codes for the 16 dinucleotide sequences showed that neither the NtC class AA00, which forms the scaffold of RNA structures, nor BB00, the DNA most populated class, are sequence neutral but their distributions are significantly biased. The reported automated assignment of the NtC classes and CANA codes available at dnatco.org provides a powerful tool for unbiased analysis of nucleic acid structures by structural and molecular biologists.

Integrated analysis sheds light on evolutionary trajectories of young transcription start sites in the human genome.

Understanding the molecular mechanisms and evolution of the gene regulatory system remains a major challenge in biology. Transcription start sites (TSSs) are especially interesting because they are central to initiating gene expression. Previous studies revealed widespread transcription initiation and fast turnover of TSSs in mammalian genomes. Yet, how new TSSs originate and how they evolve over time remain poorly understood. To address these questions, we analyzed ∼200,000 human TSSs by integrating evolutionary (inter- and intra-species) and functional genomic data, particularly focusing on evolutionarily young TSSs that emerged in the primate lineage. TSSs were grouped according to their evolutionary age using sequence alignment information as a proxy. Comparisons of young and old TSSs revealed that (1) new TSSs emerge through a combination of intrinsic factors, like the sequence properties of transposable elements and tandem repeats, and extrinsic factors such as their proximity to existing regulatory modules; (2) new TSSs undergo rapid evolution that reduces the inherent instability of repeat sequences associated with a high propensity of TSS emergence; and (3) once established, the transcriptional competence of surviving TSSs is gradually enhanced, with evolutionary changes subject to temporal (fewer regulatory changes in younger TSSs) and spatial constraints (fewer regulatory changes in more isolated TSSs). These findings advance our understanding of how regulatory innovations arise in the genome throughout evolution and highlight the genomic robustness and evolvability in these processes.

PlasClass improves plasmid sequence classification.

Many bacteria contain plasmids, but separating between contigs that originate on the plasmid and those that are part of the bacterial genome can be difficult. This is especially true in metagenomic assembly, which yields many contigs of unknown origin. Existing tools for classifying sequences of plasmid origin give less reliable results for shorter sequences, are trained using a fraction of the known plasmids, and can be difficult to use in practice. We present PlasClass, a new plasmid classifier. It uses a set of standard classifiers trained on the most current set of known plasmid sequences for different sequence lengths. We tested PlasClass sequence classification on held-out data and simulations, as well as publicly available bacterial isolates and plasmidome samples and plasmids assembled from metagenomic samples. PlasClass outperforms the state-of-the-art plasmid classification tool on shorter sequences, which constitute the majority of assembly contigs, allowing it to achieve higher F1 scores in classifying sequences from a wide range of datasets. PlasClass also uses significantly less time and memory. PlasClass can be used to easily classify plasmid and bacterial genome sequences in metagenomic or isolate assemblies. It is available under the MIT license from: https://github.com/Shamir-Lab/PlasClass.

Comparative analysis of chicken cecal microbial diversity and taxonomic composition in response to dietary variation using 16S rRNA amplicon sequencing.

BACKGROUND: Antibiotic resistance poses a grave threat to One-Health. By replacing antibiotics with non-antibiotic additives (are alternatives to antibiotics, ATAs) like phytogenic feed additives and organic acids in poultry feed. ATAs are a potential alternative as these decline the proliferation of pathogenic bacteria and strengthen gut function in broiler chickens. In this study, we use 16S rRNA amplicon sequencing of the V3-V4 region to evaluate phytogenic feed additives and organic acids on the cecal microbial diversity of broiler chickens. METHODS AND RESULTS: Two hundred & forty broiler chicks were divided into five treatments comprising: a controlled basal diet (CON), antibiotic group (AB), phytogenic feed additives (PHY), organic acids (ORG), and a combination of PHY + ORG (COM). A distinctive microbial community structure was observed amongst different treatments with increased microbial diversity in AB, ORG, and COM (p < 0.05). The synergistic effects of PHY and ORG increased bacterial population of phyla: Firmicutes, Bacteroides, and Proteobacteria in the cecum. The presence of species, Akkermansia muciniphila (involved in mucin degradation) and Bacillus safensis (a probiotic bacterium) were noticed in COM and PHY, respectively. Clustering analysis revealed a higher relative abundance of similar microbial community composition between AB and ORG groups. CONCLUSIONS: Treatments with PHY and ORG modified the relative abundance and presence/absence of specific microbiota in the chicken cecum. Hence, cecal microbiota modulation through diet is a promising strategy to reduce cross-contamination of zoonotic poultry pathogens, led to healthy and economical broiler meat.

Predictors of maternal-origin microchimerism in young women in the Philippines.

OBJECTIVES: Microchimerism is the presence of a small quantity of cells or DNA from a genetically distinct individual. This phenomenon occurs with bidirectional maternal-fetal exchange during pregnancy. Microchimerism can persist for decades after delivery and have long-term health implications. However, little is known about why microchimerism is detectable at varying levels in different individuals. We examine the variability and the following potential determinants of maternal-origin microchimerism (MMc) in young women in the Philippines: gestational duration (in utero exposure to MMc), history of being breastfed (postpartum exposure to MMc), maternal telomere length (maternal cells' ability to replicate and persist), and participant's pregnancies in young adulthood (effect of adding fetal-origin microchimerism to preexisting MMc). MATERIALS AND METHODS: Data are from the Cebu Longitudinal Health and Nutrition Survey, a population-based study of infant feeding practices and long-term health outcomes. We quantified MMc using quantitative PCR (qPCR) in 89 female participants, ages 20-22, and analyzed these data using negative binomial regression. RESULTS: In a multivariate model including all predictors, being breastfed substantially predicted decreased MMc (detection rate ratio = 0.15, p = 0.007), and there was a trend of decreasing MMc in participants who had experienced more pregnancies (detection rate ratio = 0.55, p = 0.057). DISCUSSION: These results might be explained by breastfeeding having lasting impact on immune regulatory networks, thus reducing MMc persistence. MMc may also decrease in response to the introduction of fetal-origin microchimerism with pregnancies experienced in adulthood.

Targeting individual cells by barcode in pooled sequence libraries.

Transcriptional profiling of thousands of single cells in parallel by RNA-seq is now routine. However, due to reliance on pooled library preparation, targeting analysis to particular cells of interest is difficult. Here, we present a multiplexed PCR method for targeted sequencing of select cells from pooled single-cell sequence libraries. We demonstrated this molecular enrichment method on multiple cell types within pooled single-cell RNA-seq libraries produced from primary human blood cells. We show how molecular enrichment can be combined with FACS to efficiently target ultra-rare cell types, such as the recently identified AXL+SIGLEC6+ dendritic cell (AS DC) subset, in order to reduce the required sequencing effort to profile single cells by 100-fold. Our results demonstrate that DNA barcodes identifying cells within pooled sequencing libraries can be used as targets to enrich for specific molecules of interest, for example reads from a set of target cells.

Fertility care in the era of commercial direct-to-consumer home DNA kits: issues to ponder.

Recently, we have witnessed a shift in the nature of genetic testing. What was once solely in the hands of the scientific community is now easily accessible to anyone. Commercial companies such as Veritas Genetics, Ancestry, and 23andMe offer cheap direct-to-consumer home DNA kits that are branded as a "health and ancestry service." These tests are gaining in popularity, and it is estimated that since their marketing, over 30 million people worldwide have provided their DNA samples. The implications of this new genetic era are diverse, ranging from the individuals personal health assessment, to family genealogy and ancestry, and the complexity of establishing an enormous population-based genetic database. Unique implications of commercial DNA tests on fertility patients and heath care providers, such as in the case of gamete donation, are important to acknowledge and have implications for reproductive care.

Development of a novel multiplex polymerase chain reaction system for forensic individual identification using insertion/deletion polymorphisms.

Insertion/deletion (InDel) polymorphisms have been widely used in the fields of population genetics, genetic map constructions, and forensic investigations owing to the advantages of their low mutation rates, widespread distributions in the human genome, and small amplicon sizes. In order to provide more InDels with high discrimination power in Chinese populations, we selected and constructed one novel multiplex PCR-InDel panel for forensic individual identification. Genetic distributions of these 35 InDels in five reference populations from East Asia showed low genetic differentiations among these populations. Forensic efficiency evaluations of these InDels revealed that these loci could perform well for forensic individual identifications in these reference populations. In the meantime, genetic diversities and forensic parameters of these InDels were further investigated in the studied Kazak group. Mean value of polymorphism information content for 35 InDels was 0.3611. Cumulative power of discrimination of 35 InDels was 0.99999999999999603 in Kazak group. Given these results, the panel is suitable for individual identifications in the studied Kazak and these reference populations.

Developmental validation of the Microreader™ 20A ID system.

The Microreader™ 20A ID system is designed for forensic applications such as personal identification, parentage testing, and research. It includes 13 combined DNA index system (CODIS) short tandem repeat (STR) loci (CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11), three expanded CODIS STR loci (D12S391, D19S433, and D2S1338), three non-CODIS STR loci (D6S1043, Penta D, and Penta E), and the amelogenin locus in one reaction with a six-dye fluorescent (FAM, HEX, TAMAR, ROX, PUR, and QD550) analysis system. In this study, the Microreader™ 20A ID system was validated according to the Scientific Working Group on DNA Analysis Methods validation guidelines for forensic DNA Analysis methods and Chinese national standard, including PCR-based studies, sensitivity study, precision, and accuracy evaluation, stutter calculation, inhibitor tests, species specificity, and DNA mixture studies. Our results suggest that the Microreader™ 20A ID system is a useful tool for personal identification and parentage testing.

Development of a new 25plex STRs typing system for forensic application.

We have developed a novel STR 25-plex florescence multiplex-STR kit (DNATyper25) to genotype 23 autosomal and two sex-linked loci for forensic applications and paternity analysis. Of the 23 autosomal loci, 20 are non-CODIS. The sex-linked markers include a Y-STR locus (DYS391) and the Amelogenin gene. We present developmental validation studies to show that the DNATyper25 kit is reproducible, accurate, sensitive, and robust. Sensitivity testing showed that full profiles were achieved with as low as 125 pg of human DNA. Specificity testing demonstrated a lack of cross reactivity with a variety of commonly encountered non-human DNA contaminants. Stability testing showed that full profiles were obtained with humic acid concentration ≤60 ng/µL and hematin concentration <400 µM. For forensic evaluation, the 23 autosomal STRs followed the Hardy-Weinberg equilibrium. In an analysis of 509 Chinese (CN) Hans, we detected a combined total of 181 alleles at the 23 autosomal STR loci. Since these autosomal STRs are independent from one another, PM was 8.4528 × 10-22 , TDP was 0.999 999 999 999 999 999 999, CEP was 0.999 999 8395. The forensic efficiency parameters demonstrated that these autosomal STRs are highly polymorphic and informative in the Han population of China. We performed population comparisons and showed that the Northern CN Han has a close genetic relationship with the Luzhou Han, Tujia, and Bai populations. We propose that the DNATyper25 kit will be useful for cases where paternity analysis is difficult and for situations where DNA samples are limited in quantity and low in quality.

Diversity and biogeography of frogs in the genus Amnirana (Anura: Ranidae) across sub-Saharan Africa.

Frogs in the genus Amnirana (family Ranidae) are widely distributed across sub-Saharan Africa and present a model system for exploring the relationship between diversification and geography across the continent. Using multiple loci from the mitochondrial (16S) and nuclear genomes (DISP2, FICD, KIAA2013, REV3L), we generated a strongly supported species-level phylogeny that provides insights into the continental biogeography of African species of Amnirana, which form a monophyletic group within the genus. Species delimitation analyses suggest that there may be as many as seven additional species of Amnirana in Africa. The biogeographic history of Amnirana is marked by several dispersal and vicariance events, including dispersal from the Lower Guinean Forest into the Congo Basin. In addition, phylogeographic patterns within two widespread species, A. albolabris and A. galamensis, reveal undescribed cryptic diversity. Populations assigned to A. albolabris in western Africa are more closely related to A. fonensis and require recognition as a distinct species. Our analyses reveal that the Lower and Upper Guinean Forest regions served as important centers of interspecific and intraspecific diversifications for Amnirana.

Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype.

Quantification of cell-free DNA (cfDNA) in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD) quantifies donor-derived cfDNA (dd-cfDNA) by taking advantage of single-nucleotide polymorphisms (SNPs) distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM) of identity-by-descent (IBD) states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD). These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

The Dfam database of repetitive DNA families.

Repetitive DNA, especially that due to transposable elements (TEs), makes up a large fraction of many genomes. Dfam is an open access database of families of repetitive DNA elements, in which each family is represented by a multiple sequence alignment and a profile hidden Markov model (HMM). The initial release of Dfam, featured in the 2013 NAR Database Issue, contained 1143 families of repetitive elements found in humans, and was used to produce more than 100 Mb of additional annotation of TE-derived regions in the human genome, with improved speed. Here, we describe recent advances, most notably expansion to 4150 total families including a comprehensive set of known repeat families from four new organisms (mouse, zebrafish, fly and nematode). We describe improvements to coverage, and to our methods for identifying and reducing false annotation. We also describe updates to the website interface. The Dfam website has moved to http://dfam.org. Seed alignments, profile HMMs, hit lists and other underlying data are available for download.

New thoughts on an old riddle: What determines genetic diversity within and between species?

The question of what determines genetic diversity has long remained unsolved by the modern evolutionary theory (MET). However, it has not deterred researchers from producing interpretations of genetic diversity by using MET. We examine the two observations of genetic diversity made in the 1960s that contributed to the development of MET. The interpretations of these observations by MET are widely known to be inadequate. We review the recent progress of an alternative framework, the maximum genetic diversity (MGD) hypothesis, that uses axioms and natural selection to explain the vast majority of genetic diversity as being at equilibrium that is largely determined by organismal complexity. The MGD hypothesis absorbs the proven virtues of MET and considers its assumptions relevant only to a much more limited scope. This new synthesis has accounted for the overlooked phenomenon of progression towards higher complexity, and more importantly, been instrumental in directing productive research.

Development of a rapid 21-plex autosomal STR typing system for forensic applications.

DNA-STR genotyping technology has been widely used in forensic investigations. Even with such success, there is a great need to reduce the analysis time. In this study, we established a new rapid 21-plex STR typing system, including 13 CODIS loci, Penta D, Penta E, D12S391, D2S1338, D6S1043, D19S433, D2S441 and Amelogenin loci. This system could shorten the amplification time to a minimum of 90 min and does not require DNA extraction from the samples. Validation of the typing system complied with the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese National Standard (GA/T815-2009) guidelines. The results demonstrated that this 21-plex STR typing system was a valuable tool for rapid criminal investigation.

Fishing in the Water: Effect of Sampled Water Volume on Environmental DNA-Based Detection of Macroinvertebrates.

Accurate detection of organisms is crucial for the effective management of threatened and invasive species because false detections directly affect the implementation of management actions. The use of environmental DNA (eDNA) as a species detection tool is in a rapid development stage; however, concerns about accurate detections using eDNA have been raised. We evaluated the effect of sampled water volume (0.25 to 2 L) on the detection rate for three macroinvertebrate species. Additionally, we tested (depending on the sampled water volume) what amount of total extracted DNA should be screened to reduce uncertainty in detections. We found that all three species were detected in all volumes of water. Surprisingly, however, only one species had a positive relationship between an increased sample volume and an increase in the detection rate. We conclude that the optimal sample volume might depend on the species-habitat combination and should be tested for the system where management actions are warranted. Nevertheless, we minimally recommend sampling water volumes of 1 L and screening at least 14 µL of extracted eDNA for each sample to reduce uncertainty in detections when studying macroinvertebrates in rivers and using our molecular workflow.

[Detection of Babesia canis (Piroplasmida) DNA in the blood samples and lysates of the ticks Dermacentor reticulatus (Ixodidae) collected in the Tula and Moscow Regions].

Chimeric primers, the sensitivity and specificity of which allow them to be used in both the clinical setting and the epizootological assessment of tick infection by a real-time polymerase chain reaction (RT-PCR) assay, have been designed against Babesia canis infection. The findings suggest that a large number of Babesia DNA copies are detectable in the blood in acute babesiosis. Some animals that had experienced babesiosis developed blood B. canis carriage--a small number oftrophozoites remained alive for a long time. When babesiosis was suspected, its diagnosis could be confirmed by RT-PCR in half of dogs with subclinical signs. The tick concentration of Babesia ranged from several hundred to a few thousand parasites. There were no significant differences in the number of Babesia parasites in the infected ticks in relation to their collection site. However, the occurrence of infected ticks was significantly higher in the places of constant contact with a canine population, which is indicative of the decisive role of dogs in the intensity of an epizootic process in the foci of B. canis infection.

Assessment of the purity of isolated cell populations for lineage-specific chimerism monitoring post haematopoietic stem cell transplantation.

Following haematopoietic stem cell transplantation, monitoring the proportion of donor and recipient haematopoiesis in the patient (chimerism) is an influential tool in directing further treatment choices. Short tandem repeat (STR) analysis is a method of chimerism monitoring using DNA isolated from peripheral blood, bone marrow or specific isolated cell lineages such as CD3+ T cells. For lineage-specific STR analysis on cell populations isolated from peripheral blood, a qualitative estimation of the purity of each isolated population is essential for the correct interpretation of the test data. We describe a rapid, inexpensive method for the determination of purity using a simple flow cytometry method. The method described for assessing the purity of sorted CD3+ cells can be applied to any cell population isolated using the same technology. Data obtained were comparable to results from a commercial polymerase chain reaction (PCR)-based method for the assessment of purity (Non-T Genomic Detection Kit, Accumol, Calgary, AB, Canada) (P = 0.59). Of the 303 samples tested by flow cytometry, 290 (95.7%) exceeded 90% purity, and 215 (70.95%) were over 99% pure. There were some outlying samples, showing diversity between samples and the unpredictability of purity of isolated cell populations. This flow cytometry method can be easily assimilated into routine testing protocols, allowing purity assessment in multiple-sorted cell populations for lineage-specific chimerism monitoring using a single secondary antibody and giving results comparable to a PCR-based method. As purity of isolated cell lineages is affected by time after venepuncture and storage temperature, assessment of each sample is recommended to give a reliable indication of sample quality and confidence in the interpretation of the results.