DNA Reads Research Articles

Bacterial DNA metabolism analysis by metagenomic next-generation sequencing (mNGS) after treatment of bloodstream infection

BackgroundWith the advent of metagenomic next-generation sequencing (mNGS), the time of DNA metabolism can be explored after bacteria be killed. In this study, we applied mNGS in investigation of the clearance profile of circulating bacteria DNA.MethodsAll of the rabbits were injected with the inactivated Escherichia coli. Using mNGS, we analyzed serial samples of plasma collected from rabbits to detect clearance profile of circulating E. coli DNA.ResultsIn this study, we found that the of E. coli DNA could still be detected 6 h after injecting killed bacteria. The clearance half-lives associated with the 2 phases are 0.37 and 1.81 h. We also explored there is no correlation between the disease severity with the E. coli DNA reads in circulation.ConclusionsAfter the bacteria were completely killed, their DNA could still be detected in the blood circulation. The metabolism of bacterial DNA in the circulation had two phases: fast and slow phases, and there were no correlations between the level of bacteria reads with the severity of patients’ disease after the bacteria have been completely killed.

Analysis of Purity and Concentration of DNA Isolated in Dragonfly (Onychogomphus forcipatus)

Analysis of the purity and concentration of DNA isolation results was carried out using samples of dragonflies (Onychogomphus forcipatus) species of insects. This research is important because there are many kinds of research in the molecular field that require good DNA isolation techniques to support the success of the research. The purpose of this paper is to provide information about the quality of the isolated DNA seen using the purity and concentration parameters. The extraction method uses the centrifuge column method. The isolated data were analyzed statistically using the average test. The results of DNA isolation showed that the average concentration value was in the range of 60.10 – 69.95. The analysis of the purity of the isolated DNA read at the A260/A280 ratio was in the range of 1.817 – 1.929, while the purity analysis at the A260/A230 ratio was in the range of 0.760 – 0.822. Based on the results of the study, it can be concluded that the analysis of the purity and concentration of DNA from the isolation of dragonfly samples was in a good category at the A260/A280 ratio, while the purity read at the A260/A230 ratio was below the value categorized as good.

Accelerated nanopore basecalling with SLOW5 data format.

Nanopore sequencing is emerging as a key pillar in the genomic technology landscape but computational constraints limiting its scalability remain to be overcome. The translation of raw current signal data into DNA or RNA sequence reads, known as 'basecalling', is a major friction in any nanopore sequencing workflow. Here, we exploit the advantages of the recently developed signal data format 'SLOW5' to streamline and accelerate nanopore basecalling on high-performance computing (HPC) and cloud environments. SLOW5 permits highly efficient sequential data access, eliminating a potential analysis bottleneck. To take advantage of this, we introduce Buttery-eel, an open-source wrapper for Oxford Nanopore's Guppy basecaller that enables SLOW5 data access, resulting in performance improvements that are essential for scalable, affordable basecalling. Buttery-eel is available at https://github.com/Psy-Fer/buttery-eel.

Calibration of cell-free DNA measurements by next-generation sequencing.

Accurate monitoring of disease burden depends on accurate disease marker quantification. Although next-generation sequencing (NGS) is a promising technology for noninvasive monitoring, plasma cell-free DNA levels are often reported in misleading units that are confounded by non-disease-related factors. We proposed a novel strategy for calibrating NGS assays using spiked normalizers to improve precision and to promote standardization and harmonization of analyte concentrations. In this study, we refined our NGS protocol to calculate absolute analyte concentrations to (1) adjust for assay efficiency, as judged by recovery of spiked synthetic normalizer DNAs, and (2) calibrate NGS values against droplet digital polymerase chain reaction (ddPCR). As a model target, we chose the Epstein-Barr virus (EBV) genome. In patient (n = 12) and mock (n = 12) plasmas, NGS and 2 EBV ddPCR assays were used to report EBV load in copies per mL of plasma. Next-generation sequencing was equally sensitive to ddPCR, with improved linearity when NGS values were normalized for spiked DNA read counts (R2 = 0.95 for normalized vs 0.91 for raw read concentrations). Linearity permitted NGS calibration to each ddPCR assay, achieving equivalent concentrations (copies/mL). Our novel strategy for calibrating NGS assays suggests potential for a universal reference material to overcome biological and preanalytical variables hindering traditional NGS strategies for quantifying disease burden.

You are what you excrete.

Residual human DNA reads in sequenced faecal samples may allow some individuals to be identified.

Efficient short read mapping to a pangenome that is represented by a graph of ED strings.

A pangenome represents many diverse genome sequences of the same species. In order to cope with small variations as well as structural variations, recent research focused on the development of graph-based models of pangenomes. Mapping is the process of finding the original location of a DNA read in a reference sequence, typically a genome. Using a pangenome instead of a (linear) reference genome can, e.g. reduce mapping bias, the tendency to incorrectly map sequences that differ from the reference genome. Mapping reads to a graph, however, is more complex and needs more resources than mapping to a reference genome. Reducing the complexity of the graph by encoding simple variations like SNPs in a simple way can accelerate read mapping and reduce the memory requirements at the same time. We introduce graphs based on elastic-degenerate strings (ED strings, EDS) and the linearized form of these EDS graphs as a new representation for pangenomes. In this representation, small variations are encoded directly in the sequence. Structural variations are encoded in a graph structure. This reduces the size of the representation in comparison to sequence graphs. In the linearized form, mapping techniques that are known from ordinary strings can be applied with appropriate adjustments. Since most variations are expressed directly in the sequence, the mapping process rarely has to take edges of the EDS graph into account. We developed a prototypical software tool GED-MAP that uses this representation together with a minimizer index to map short reads to the pangenome. Our experiments show that the new method works on a whole human genome scale, taking structural variants properly into account. The advantage of GED-MAP, compared with other pangenomic short read mappers, is that the new representation allows for a simple indexing method. This makes GED-MAP fast and memory efficient. Sources are available at: https://github.com/thomas-buechler-ulm/gedmap.

Pan-Arctic plankton community structure and its global connectivity

The Arctic Ocean (AO) is being rapidly transformed by global warming, but its biodiversity remains understudied for many planktonic organisms, in particular for unicellular eukaryotes that play pivotal roles in marine food webs and biogeochemical cycles. The aim of this study was to characterize the biogeographic ranges of species that comprise the contemporary pool of unicellular eukaryotes in the AO as a first step toward understanding mechanisms that structure these communities and identifying potential target species for monitoring. Leveraging the Tara Oceans DNA metabarcoding data, we mapped the global distributions of operational taxonomic units (OTUs) found on Arctic shelves into five biogeographic categories, identified biogeographic indicators, and inferred the degree to which AO communities of unicellular eukaryotes share members with assemblages from lower latitudes. Arctic/Polar indicator OTUs, as well as some globally ubiquitous OTUs, dominated the detection and abundance of DNA reads in the Arctic samples. OTUs detected only in Arctic samples (Arctic-exclusives) showed restricted distribution with relatively low abundances, accounting for 10–16% of the total Arctic OTU pool. OTUs with high abundances in tropical and/or temperate latitudes (non-Polar indicators) were also found in the AO but mainly at its periphery. We observed a large change in community taxonomic composition across the Atlantic-Arctic continuum, supporting the idea that advection and environmental filtering are important processes that shape plankton assemblages in the AO. Altogether, this study highlights the connectivity between the AO and other oceans, and provides a framework for monitoring and assessing future changes in this vulnerable ecosystem.

Genomics, Population Divergence, and Historical Demography of the World's Largest and Endangered Butterfly, The Queen Alexandra's Birdwing.

The world's largest butterfly is the microendemic Papua New Guinean Ornithoptera alexandrae. Despite years of conservation efforts to protect its habitat and breed this up-to-28-cm butterfly, this species still figures as endangered in the IUCN Red List and is only known from two allopatric populations occupying a total of only ∼140 km². Here we aim at assembling reference genomes for this species to investigate its genomic diversity, historical demography and determine whether the population is structured, which could provide guidance for conservation programs attempting to (inter)breed the two populations. Using a combination of long and short DNA reads and RNA sequencing, we assembled six reference genomes of the tribe Troidini, with four annotated genomes of O. alexandrae and two genomes of related species Ornithoptera priamus and Troides oblongomaculatus. We estimated the genomic diversity of the three species, and we proposed scenarios for the historical population demography using two polymorphism-based methods taking into account the characteristics of low-polymorphic invertebrates. Indeed, chromosome-scale assemblies reveal very low levels of nuclear heterozygosity across Troidini, which appears to be exceptionally low for O. alexandrae (lower than 0.01%). Demographic analyses demonstrate low and steadily declining Ne throughout O. alexandrae history, with a divergence into two distinct populations about 10,000 years ago. These results suggest that O. alexandrae distribution has been microendemic for a long time. It should also make local conservation programs aware of the genomic divergence of the two populations, which should not be ignored if any attempt is made to cross the two populations.

Identifying Taxonomic Units in Metagenomic DNA Streams on Mobile Devices.

With the emergence of portable DNA sequencers, such as Oxford Nanopore Technology MinION, metagenomic DNA sequencing can be performed in real-time and directly in the field. However, because metagenomic DNA analysis tasks, e.g., classification, taxonomic units assignment, etc., are compute and memory intensive, and the available methods are designed for batch processing, the current metagenomic tools are not well suited for mobile devices. In this work, we propose a new memory-efficient approach to identify Operational Taxonomic Units (OTUs) in metagenomic DNA streams on mobile devices. Our method is based on finding connected components in overlap graphs constructed over a real-time stream of long DNA reads as produced by the MinION platform. We propose an efficient algorithm to maintain connected components when an overlap graph is streamed and show how redundant information can be removed from the stream by transitive closures. We also propose how our algorithms can be integrated into a larger DNA analysis pipeline tailored for mobile computing. Through experiments on simulated and real-world metagenomic data, executed on the actual mobile device, we demonstrate that our resulting solution is able to recover OTUs with high precision. Our experiments also demonstrate the compounding benefits of introducing feedback loops in the DNA analysis pipeline.

ArcHives—combined palynological, genomic and lipid analysis of medieval wax seals

Beeswax is a product of honeybees (Apis mellifera) and has been used extensively through time, especially as the primary component in medieval sealing wax for authenticating millions of documents. Today, these seals form large collections which, along with the historical information in the documents that the seals are attached to, could be a potential biomolecular archive for honeybees. Here, we investigate the possibility of obtaining biological information from medieval wax seals by performing a palynological and shotgun metagenomic analysis on eight medieval wax seal fragments. Our palynological results show that some pollen and fungal spores remain in the seals, albeit very little. Only one out of eight samples yielded enough DNA for sequencing. Moreover, only minor parts of the DNA reads could be taxonomically identified and were identified as plant and fungal DNA. These results demonstrate some potential for using wax seals as biological archives, but most importantly provides a framework for future studies, in addition to understanding further the degradation of seals as cultural heritage objects. We emphasize that future analyses should focus on other methodologies to retrieve data for historical context or alternatively improve molecular methods and screen sample collections broadly.

Analysis of Tandem Repeat Expansions Using Long DNA Reads.

Abnormal expansion or shortening of tandem repeats can cause a variety of genetic diseases. The use of long DNA reads has facilitated the analysis of disease-causing repeats in the human genome. Long read sequencers enable us to directly analyze repeat length and sequence content by covering whole repeats; they are therefore considered suitable for the analysis of long tandem repeats. Here, we describe an expanded repeat analysis using target sequencing data produced by the Oxford Nanopore Technologies (hereafter referred to as ONT) nanopore sequencer.

Generative Adversarial Networks for DNA Storage Channel Simulator

DNA data storage systems have rapidly developed with novel error-correcting techniques, random access algorithms, and query systems. However, designing an algorithm for DNA storage systems is challenging, mainly due to the unpredictable nature of errors and the extremely high price of experiments. Thus, a simulator is of interest that can imitate the error statistics of a DNA storage system and replace the experiments in developing processes. We introduce novel generative adversarial networks that learn DNA storage channel statistics. Our simulator takes oligos (DNA sequences to write) as an input and generates a FASTQ file that includes output DNA reads and quality scores as if the oligos are synthesized and sequenced. We trained the proposed simulator with data from a single experiment consisting of 14,400 input oligo strands and 12,108,573 output reads. The error statistics between the input and the output of the trained generator match the actual error statistics, including the error rate at each position, the number of errors for each nucleotide, and high-order statistics. The code is available at <monospace xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><uri>https://github.com/gyfbianhuanyun/DNA_storage_simulator_GAN</uri></monospace> .

Genetic and phenotypic associations of mitochondrial DNA copy number, SNP, and haplogroups with growth and carcass traits in beef cattle

Mitochondrial DNA copy number (mtDNA CN) is heritable and easily obtained from low-pass sequencing (LPS). This study investigated the genetic correlation of mtDNA CN with growth and carcass traits in a multi-breed and crossbred beef cattle population. Blood, leucocyte, and semen samples were obtained from 2,371 animals and subjected to LPS that resulted in nuclear DNA (nuDNA) and mtDNA sequence reads. Mitochondrial DNA CN was estimated as the ratio of mtDNA to nuDNA coverages. Variant calling was performed from mtDNA, and 11 single nucleotide polymorphisms (SNP) were identified in the population. Samples were classified in taurine haplogroups. Haplogroup and mtDNA type were further classified based on the 11 segregating SNP. Growth and carcass traits were available for between 7,249 and 60,989 individuals. Associations of mtDNA CN, mtDNA haplogroups, mtDNA types, and mtDNA SNP with growth and carcass traits were estimated with univariate animal models, and genetic correlations were estimated with a bivariate animal model based on pedigree. Mitochondrial DNA CN tended (P-value ≤0.08) to be associated with birth weight and weaning weight. There was no association (P-value >0.10) between mtDNA SNP, haplogroups, or types with growth and carcass traits. Genetic correlation estimates of mtDNA CN were -0.30 ± 0.16 with birth weight, -0.31 ± 0.16 with weaning weight, -0.15 ± 0.14 with post-weaning gain, -0.11 ± 0.19 with average daily dry-matter intake, -0.04 ± 0.22 with average daily gain, -0.29 ± 0.13 with mature cow weight, -0.11 ± 0.13 with slaughter weight, -0.14 ± 0.13 with carcass weight, -0.07 ± 0.14 with carcass backfat, 0.14 ± 0.14 with carcass marbling, and -0.06 ± 0.14 with ribeye area. In conclusion, mtDNA CN was negatively correlated with most traits investigated, and the genetic correlation was stronger with growth traits than with carcass traits.

Is endozoochoric seed dispersal by large herbivores an evolutionary adaptation? Revisiting the Janzen's ‘Foliage is the fruit’ hypothesis

Many plant species develop fruits to attract animals that will eat them and then disperse the seeds. However, there are many plant species, whose seeds are dispersed endozoochorically, but their fruits are not particularly attractive to animals. The “Foliage is the fruit” (FF) hypothesis proposes that entire biomass of the plant exists to encourage herbivores to eat it, in order to enhance seed intake and dispersal (Janzen, 1984).We tested the FF hypothesis by combining the results from the greenhouse seedling emergence method (GR) and DNA-metabarcoding of plant remnants in faeces of European moose (Alces alces L.). We processed 665 samples by the GR and 429 by the MB method, hypothesizing that if the safe passage of seeds through the gut of a large herbivore is the result of an evolutionary adaptation to endozoochoric dispersal, then the species composition of plants revealed by the two methods should largely overlap and the abundance of seedlings revealed by the GR method should be positively correlated in time with the read abundance of DNA of the same species. The large discrepancy between the lists of species detected by DNA metabarcoding and the GR method argues against the FF hypothesis. However, in the case of Urtica dioica, Lysimachia vulgaris and Lythrum salicaria, some clues of evolutionary adaptation to endozoochoric dispersal were revealed for: 1) their foliage is attractive to herbivores; 2) seeds are small, rounded in shape, yielded in large numbers and pass safely through the herbivore's gut; 3) the abundance of seeds (seedlings) was significantly and strongly influenced by the abundance of the plant biomass (DNA reads) in dung samples; 4) peaks in seed abundance and biomass consumption coincided in time. However, it should be considered that moose's diet is mostly composed of woody browse, which makes this animal not an optimal model for testing the Janzen's hypothesis and studies on typical grazers are needed in this respect.

Fungal and fungal-like diversity in marine sediments from the maritime Antarctic assessed using DNA metabarcoding

We assessed the fungal and fungal-like sequence diversity present in marine sediments obtained in the vicinity of the South Shetland Islands (Southern Ocean) using DNA metabarcoding through high-throughput sequencing (HTS). A total of 193,436 DNA reads were detected in sediment obtained from three locations: Walker Bay (Livingston Island) at 52 m depth (48,112 reads), Whalers Bay (Deception Island) at 151 m (104,704) and English Strait at 404 m (40,620). The DNA sequence reads were assigned to 133 distinct fungal amplicon sequence variants (ASVs) representing the phyla Ascomycota, Basidiomycota, Mortierellomycota, Chytridiomycota, Glomeromycota, Monoblepharomycota, Mucoromycota and Rozellomycota and the fungal-like Straminopila. Thelebolus balaustiformis, Pseudogymnoascus sp., Fungi sp. 1, Ciliophora sp., Agaricomycetes sp. and Chaetoceros sp. were the dominant assigned taxa. Thirty-eight fungal ASVs could only be assigned to higher taxonomic levels, and may represent taxa not currently included in the available databases or represent new taxa and/or new records for Antarctica. The total fungal community displayed high indices of diversity, richness and moderate to low dominance. However, diversity and taxa distribution varied across the three sampling sites. In Walker Bay, unidentified fungi were dominant in the sequence assemblage. Whalers Bay sediment was dominated by Antarctic endemic and cold-adapted taxa. Sediment from English Strait was dominated by Ciliophora sp. and Chaetoceros sp. These fungal assemblages were dominated by saprotrophic, plant and animal pathogenic and symbiotic taxa. The detection of an apparently rich and diverse fungal community in these marine sediments reinforces the need for further studies to characterize their richness, functional ecology and potential biotechnological applications.

Fine-scale diversity of prey detected in humpback whale feces.

Predator diets are largely influenced by prey availability and abundance. Yet, in heterogenous marine environments, identifying the prey species consumed by diving mammals remains a fundamental challenge. For rorqual whales, the energetic constraints of prey engulfment require that the whales seek areas of high prey abundance and execute discrete lunge feeding events on patches of high-density prey. Prey occurrences in feces should therefore provide meaningful insight into the dominant taxa in food patches selected by the animal. We investigated the prey consumed by humpback whales in three regions in southern British Columbia (BC), Canada, using opportunistic fecal sampling, microscopy, and DNA metabarcoding of 14 fecal samples. Fish including Pacific herring (Clupea pallasii), hake (Merluccius productus), and eulachon (Thaleichthys pacificus) were the most common fish species potentially targeted by humpback whales in two regions. The krill Euphausia pacifica was the most prevalent invertebrate DNA detected in all three regions, while sergestid and mysid shrimp may also be important. High DNA read abundances from walleye pollock (Gadus chalcogrammus) and sablefish (Anoplopoma fimbria) were also recovered in one sample each, suggesting that juveniles of these semi-pelagic species may occasionally be targeted. In general, we observed heavily digested fecal material that drove substantial dissimilarities in taxonomic resolution between polymerase chain reaction-based and morphological analyses of the feces. Pacific herring and walleye pollock were the only prey species confirmed by both methods. Our results highlight that molecular and visual analyses of fecal samples provide a complementary approach to diet analysis, with each method providing unique insight into prey diversity.

Finding Rearrangements in Nanopore DNA Reads with LAST and dnarrange.

Long-read DNA sequencing techniques such as nanopore are especially useful for characterizing complex sequence rearrangements, which occur in some genetic diseases and also during evolution. Analyzing the sequence data to understand such rearrangements is not trivial, due to sequencing error, rearrangement intricacy, and abundance of repeated similar sequences in genomes.The LAST and dnarrange software packages can resolve complex relationships between DNA sequences and characterize changes such as gene conversion, processed pseudogene insertion, and chromosome shattering. They can filter out numerous rearrangements shared by controls, e.g., healthy humans versus a patient, to focus on rearrangements unique to the patient. One useful ingredient is last-train, which learns the rates (probabilities) of deletions, insertions, and each kind of base match and mismatch. These probabilities are then used to find the most likely sequence relationships/alignments, which is especially useful for DNA with unusual rates, such as DNA from Plasmodium falciparum (malaria) with ∼80% a+t. This is also useful for less-studied species that lack reference genomes, so the DNA reads are compared to a different species' genome. We also point out that a reference genome with ancestral alleles would be ideal.

Propagation of viral genomes by replicating ammonia-oxidising archaea during soil nitrification

Ammonia-oxidising archaea (AOA) are a ubiquitous component of microbial communities and dominate the first stage of nitrification in some soils. While we are beginning to understand soil virus dynamics, we have no knowledge of the composition or activity of those infecting nitrifiers or their potential to influence processes. This study aimed to characterise viruses having infected autotrophic AOA in two nitrifying soils of contrasting pH by following transfer of assimilated CO2-derived 13C from host to virus via DNA stable-isotope probing and metagenomic analysis. Incorporation of 13C into low GC mol% AOA and virus genomes increased DNA buoyant density in CsCl gradients but resulted in co-migration with dominant non-enriched high GC mol% genomes, reducing sequencing depth and contig assembly. We therefore developed a hybrid approach where AOA and virus genomes were assembled from low buoyant density DNA with subsequent mapping of 13C isotopically enriched high buoyant density DNA reads to identify activity of AOA. Metagenome-assembled genomes were different between the two soils and represented a broad diversity of active populations. Sixty-four AOA-infecting viral operational taxonomic units (vOTUs) were identified with no clear relatedness to previously characterised prokaryote viruses. These vOTUs were also distinct between soils, with 42% enriched in 13C derived from hosts. The majority were predicted as capable of lysogeny and auxiliary metabolic genes included an AOA-specific multicopper oxidase suggesting infection may augment copper uptake essential for central metabolic functioning. These findings indicate virus infection of AOA may be a frequent process during nitrification with potential to influence host physiology and activity.

SparkEC: speeding up alignment-based DNA error correction tools

BackgroundIn recent years, huge improvements have been made in the context of sequencing genomic data under what is called Next Generation Sequencing (NGS). However, the DNA reads generated by current NGS platforms are not free of errors, which can affect the quality of downstream analysis. Although error correction can be performed as a preprocessing step to overcome this issue, it usually requires long computational times to analyze those large datasets generated nowadays through NGS. Therefore, new software capable of scaling out on a cluster of nodes with high performance is of great importance.ResultsIn this paper, we present SparkEC, a parallel tool capable of fixing those errors produced during the sequencing process. For this purpose, the algorithms proposed by the CloudEC tool, which is already proved to perform accurate corrections, have been analyzed and optimized to improve their performance by relying on the Apache Spark framework together with the introduction of other enhancements such as the usage of memory-efficient data structures and the avoidance of any input preprocessing. The experimental results have shown significant improvements in the computational times of SparkEC when compared to CloudEC for all the representative datasets and scenarios under evaluation, providing an average and maximum speedups of 4.9times and 11.9times, respectively, over its counterpart.ConclusionAs error correction can take excessive computational time, SparkEC provides a scalable solution for correcting large datasets. Due to its distributed implementation, SparkEC speed can increase with respect to the number of nodes in a cluster. Furthermore, the software is freely available under GPLv3 license and is compatible with different operating systems (Linux, Windows and macOS).

DNA read count calibration for single-molecule, long-read sequencing

There are many applications in which quantitative information about DNA mixtures with different molecular lengths is important. Gene therapy vectors are much longer than can be sequenced individually via short-read NGS. However, vector preparations may contain smaller DNAs that behave differently during sequencing. We have used two library preparations each for Pacific Biosystems (PacBio) and Oxford Nanopore Technologies NGS to determine their suitability for quantitative assessment of varying sized DNAs. Equimolar length standards were generated from E. coli genomic DNA. Both PacBio library preparations provided a consistent length dependence though with a complex pattern. This method is sufficiently sensitive that differences in genomic copy number between DNA from E. coli grown in exponential and stationary phase conditions could be detected. The transposase-based Oxford Nanopore library preparation provided a predictable length dependence, but the random sequence starts caused the loss of original length information. The ligation-based approach retained length information but read frequency was more variable. Modeling of E. coli versus lambda read frequency via cubic spline smoothing showed that the shorter genome could be used as a suitable internal spike-in for DNAs in the 200 bp to 10 kb range, allowing meaningful QC to be carried out with AAV preparations.