Read Mapping Research Articles

PSIII-6 Gene Expression Profile of Beef Heifer Placental Caruncles Is Affected by Pre-breeding and Early Gestation Micronutrient Supplementation

Abstract Vitamins and minerals are essential for proper fetal and placental development and function. However, the impact of micronutrient supplementation on placental function and gene expression remains unclear. Herein, we performed a transcriptomic analysis to determine the impact of pre-breeding maternal micronutrient supplementation on the gene expression of placental caruncles (CAR; maternal placenta). Crossbred Angus beef heifers were supplemented (VTM, n = 7) or not (CON, n = 7) with 113 g•heifer-1•d-1 of mineral premix (Purina® Wind & Rain® Storm® All-Season 7.5 Complete) from d 71 to 148 before breeding and until d 83 of gestation. After breeding, heifers were fed a diet to gain 0.79 kg/d. Uteroplacental tissues were collected at d 83. The largest placentome closest to the fetus was collected, and CAR was manually dissected from the cotyledon. Total RNA was isolated from CAR, and gene expression was measured with RNA-Seq. After data quality control and read mapping, differential expression was performed using DESeq2. We identified 46 upregulated and 19 downregulated genes in the VTM group (adj.Pval < 0.1). ShinyGO pathway analysis software was used to identify genes in the Ca and CGMP-PKG signaling pathways, including CALM2 and CAMK2G, which were down and upregulated, respectively. Calcium-mediated systems may activate steroidogenic activity in bovine placentomes, while the cGMP-PKG pathway plays a key role in vascular homeostasis mediated by nitric oxide and decreased Ca concentrations. Furthermore, biological processes underlying blood circulation were among those over-represented. Previous studies report that maternal nutrition may impact placental vascularity and uterine blood flow. ATP2B, that is upregulated in the VTM group, is a calcium/calmodulin-regulated, magnesium-dependent protein involved in intracellular Ca homeostasis. In summary, pre-breeding and early gestation maternal micronutrient supplementation leads to differential expression of genes involved in Ca homeostasis and has a putative effect on placenta vascular function.

Producing Genomic Sequences after Genome Scaffolding with Ambiguous Paths: Complexity, Approximation and Lower Bounds

Scaffolding is the final step in assembling Next Generation Sequencing data, in which pre-assembled contiguous regions (”contigs”) are oriented and ordered using information that links them (for example, mapping of paired-end reads). As the genome of some species is highly repetitive, we allow placing some contigs multiple times, thereby generalizing established computational models for this problem. We study the subsequent problems induced by the translation of solutions of the model back to actual sequences, proposing models and analyzing the complexity of the resulting computational problems. We find both polynomial-time and $$\mathcal {NP}$$ -hard special cases like planarity or bounded degree. Finally, we propose two polynomial-time approximation algorithms according to cut/weight score.

Research on the Effect of Exercise Load on Orienteering Map Reading Ability Based on Computer Software Analysis

Orienteering is a combination of physical and intellectual sports, and the combination of physiology and psychology in experimental research on orienteering is in line with the needs of the development of orienteering itself. The study of orienteering is to examine the brain’s ability to recognize maps under different exercise loads, to control the running speed, to maximize the brain’s ability to recognize maps, and to provide a reference basis for athletes, coaches, and amateur participants in orienteering to understand the relationship between running and maps. In addition, if the experimental data is collected from professional athletes, a database of physiological and biochemical indicators of our orienteering athletes can be established to improve the data reference for our orienteering training and competition. Based on this, this paper explores the effect of exercise load on orienteering map recognition ability.

In Searching Architecture and City Pattern as the Mark of Old Coastal Semarang, Indonesia

Based on the previous research on the former of Semarang capital city, Kanjengan, there is a supposition that the area is a coastal city. As the consequence of the sedimentation process occurred by Java Island, Semarang city, and some cities in Java are no longer on the seashore. The cities that are archived in the Map of Indonesia in the 15th century and some records from foreign sailors, absolutely have several different conditions compared to the past and the present condition. How the architecture and the city’s patterns of the Old Semarang? The method of this research was conducted by exploring historical articles, toponyms, maps, and old pictures which were then compared with the remaining architecture and spatial patterns. The reading of old maps and pictures was done by using the seeing by believing approach. The analysis was performed by using the manual sketch. The architectural heritage like the former lighthouse, commercial corridors in the north of the square, and toponyms were tools to prove that the area was a seaport and a waterfront city. Old Coastal Semarang has an Architecture and urban design concept as the main gate from the sea to the city center at the time. Surrounding the main gate, they were the existence of the former lighthouse (the Menara Layur Mosque), multi-ethnics houses, toponyms indicating that some places were a former harbor, and the remaining trade corridor connecting the port and the local government center. This concept is similar to the Islamic Sultanates on the north coast of Java and the waterfront-based cities in the interior of Java. It is assumed that Semarang Old city has a similar role with the other Islamic Sultanates. This area is proper to be a city conservation area and become a part that is integrated into future urban planning.

Zea mays RNA-seq estimated transcript abundances are strongly affected by read mapping bias

BackgroundGenetic variation for gene expression is a source of phenotypic variation for natural and agricultural species. The common approach to map and to quantify gene expression from genetically distinct individuals is to assign their RNA-seq reads to a single reference genome. However, RNA-seq reads from alleles dissimilar to this reference genome may fail to map correctly, causing transcript levels to be underestimated. Presently, the extent of this mapping problem is not clear, particularly in highly diverse species. We investigated if mapping bias occurred and if chromosomal features associated with mapping bias. Zea mays presents a model species to assess these questions, given it has genotypically distinct and well-studied genetic lines.ResultsIn Zea mays, the inbred B73 genome is the standard reference genome and template for RNA-seq read assignments. In the absence of mapping bias, B73 and a second inbred line, Mo17, would each have an approximately equal number of regulatory alleles that increase gene expression. Remarkably, Mo17 had 2–4 times fewer such positively acting alleles than did B73 when RNA-seq reads were aligned to the B73 reference genome. Reciprocally, over one-half of the B73 alleles that increased gene expression were not detected when reads were aligned to the Mo17 genome template. Genes at dissimilar chromosomal ends were strongly affected by mapping bias, and genes at more similar pericentromeric regions were less affected. Biased transcript estimates were higher in untranslated regions and lower in splice junctions. Bias occurred across software and alignment parameters.ConclusionsMapping bias very strongly affects gene transcript abundance estimates in maize, and bias varies across chromosomal features. Individual genome or transcriptome templates are likely necessary for accurate transcript estimation across genetically variable individuals in maize and other species.

Esoteric and exoteric vision in South Asian miniature painting

This essay focuses on contemporary artworks that hark back to Mughal examples. In particular, there is an underlying formal arrangement shared by traditional and contemporary art, which I would like to suggest has much in common with reading maps. In fact, many of these artworks actually feature maps that alert us to different ways of ‘reading’ this tradition of formalism, stimulating different levels of description.

Systematic benchmark of ancient DNA read mapping.

The current standard practice for assembling individual genomes involves mapping millions of short DNA sequences (also known as DNA 'reads') against a pre-constructed reference genome. Mapping vast amounts of short reads in a timely manner is a computationally challenging task that inevitably produces artefacts, including biases against alleles not found in the reference genome. This reference bias and other mapping artefacts are expected to be exacerbated in ancient DNA (aDNA) studies, which rely on the analysis of low quantities of damaged and very short DNA fragments (~30-80bp). Nevertheless, the current gold-standard mapping strategies for aDNA studies have effectively remained unchanged for nearly a decade, during which time new software has emerged. In this study, we used simulated aDNA reads from three different human populations to benchmark the performance of 30 distinct mapping strategies implemented across four different read mapping software-BWA-aln, BWA-mem, NovoAlign and Bowtie2-and quantified the impact of reference bias in downstream population genetic analyses. We show that specific NovoAlign, BWA-aln and BWA-mem parameterizations achieve high mapping precision with low levels of reference bias, particularly after filtering out reads with low mapping qualities. However, unbiased NovoAlign results required the use of an IUPAC reference genome. While relevant only to aDNA projects where reference population data are available, the benefit of using an IUPAC reference demonstrates the value of incorporating population genetic information into the aDNA mapping process, echoing recent results based on graph genome representations.

Strongyloides RNA-seq Browser: a web-based software platform for on-demand bioinformatics analyses of Strongyloides species.

Soil-transmitted gastrointestinal parasitic nematodes infect approximately 1 billion people worldwide, predominantly in low-resource communities. Skin-penetrating gastrointestinal nematodes in the genus Strongyloides are emerging as model systems for mechanistic studies of soil-transmitted helminths due to the growing availability of functional genomics tools for these species. To facilitate future genomics studies of Strongyloides species, we have designed a web-based application, the Strongyloides RNA-seq Browser, that provides an open source, user-friendly portal for accessing and analyzing Strongyloides genomic expression data. Specifically, the Strongyloides RNA-seq Browser takes advantage of alignment-free read mapping tools and R-based transcriptomics tools to re-analyze publicly available RNA sequencing datasets from four Strongyloides species: Strongyloides stercoralis, Strongyloides ratti, Strongyloides papillosus, and Strongyloides venezuelensis. This application permits on-demand exploration and quantification of gene expression across life stages without requiring previous coding experience. Here, we describe this interactive application and demonstrate how it may be used by nematode researchers to conduct a standard set of bioinformatics queries.

Exploring the Application of Photography in Graphic Design

With the continuous improvement of information technology, the era of map reading has come, the popularity of graphic design is getting stronger and stronger, and there are more and more integration and interlacing between different art disciplines, and photography is also involved as an important part and has played an active role in promoting the development of graphic design. The application of photography in graphic design enhances the visual attractiveness of graphic design works, making the images of works richer and the effect more intuitive and strong. The updating of the technical concept of graphic design also promotes the innovation of photography technology in the new era, and the two complement each other and improve each other, jointly promoting the development of the visual industry.

The genome of the lowland anoa (Bubalus depressicornis) illuminates the origin of river and swamp buffalo

Two types of domestic water buffalo are currently recognized: the river buffalo from the Indian subcontinent and Mediterranean countries and the swamp buffalo from China and Southeast Asia. To test the hypothesis of two separate species of water buffalo, we sequenced the genome of the lowland anoa, Bubalus depressicornis, which is a dwarf wild buffalo endemic to Sulawesi, and two genomes of swamp buffalo, and made comparisons with 12 additional genomes. Three genomic data sets were constructed to infer phylogenetic relationships: the mitochondrial genome (15,468 bp; maternal transmission), two concatenated Y-chromosomal genes, AMELY and DDX3Y (20,036 bp; paternal transmission), and a selection of 30 nuclear genes representing all cattle chromosomes (364,887 bp; biparental transmission). The comparisons between our 30 nuclear gene sequences obtained by read mapping and those directly extracted from Bos taurus and Bubalus bubalis genome assemblies show that the mapping approach revealed higher levels of heterozygosity at both nucleotide sites and indels (insertions and deletions) (0.09–0.15%), as well as several sequence errors (0.07%). Our phylogenetic and molecular dating analyses provide strong evidence that the lowland anoa, river buffalo, and swamp buffalo are three distinct taxa which separated rapidly from each other during the Pleistocene epoch. We therefore conclude that two species of domestic water buffalo should be distinguished: Bubalus bubalis for the river buffalo and Bubalus kerabau for the swamp buffalo. The new classification can have deep implications for understanding the evolution and selection of domesticated forms and for the conservation and management of wild buffalo populations in South and Southeast Asia.

Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids

BackgroundHigh-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount.MethodsPlant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis.ResultsThe study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols.ConclusionsThis study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

Demonstrating the utility of flexible sequence queries against indexed short reads with FlexTyper.

Across the life sciences, processing next generation sequencing data commonly relies upon a computationally expensive process where reads are mapped onto a reference sequence. Prior to such processing, however, there is a vast amount of information that can be ascertained from the reads, potentially obviating the need for processing, or allowing optimized mapping approaches to be deployed. Here, we present a method termed FlexTyper which facilitates a “reverse mapping” approach in which high throughput sequence queries, in the form of k-mer searches, are run against indexed short-read datasets in order to extract useful information. This reverse mapping approach enables the rapid counting of target sequences of interest. We demonstrate FlexTyper’s utility for recovering depth of coverage, and accurate genotyping of SNP sites across the human genome. We show that genotyping unmapped reads can correctly inform a sample’s population, sex, and relatedness in a family setting. Detection of pathogen sequences within RNA-seq data was sensitive and accurate, performing comparably to existing methods, but with increased flexibility. We present two examples of ways in which this flexibility allows the analysis of genome features not well-represented in a linear reference. First, we analyze contigs from African genome sequencing studies, showing how they distribute across families from three distinct populations. Second, we show how gene-marking k-mers for the killer immune receptor locus allow allele detection in a region that is challenging for standard read mapping pipelines. The future adoption of the reverse mapping approach represented by FlexTyper will be enabled by more efficient methods for FM-index generation and biology-informed collections of reference queries. In the long-term, selection of population-specific references or weighting of edges in pan-population reference genome graphs will be possible using the FlexTyper approach. FlexTyper is available at https://github.com/wassermanlab/OpenFlexTyper.

PARP1-RNA interaction analysis: PARP1 regulates the expression of extracellular matrix-related genes in HK-2 renal proximal tubular epithelial cells.

Recent studies suggest that Poly(ADP-ribose) polymerase 1 (PARP1) acts as an RNA-binding protein in a majority of renal diseases with tubular cell injury. However, detailed knowledge of RNA targets and the RNA-binding regions for PARP1 is unknown. Herein, mapping of iRIP-seq reads in HK-2 renal tubular epithelial cells showed a biased distribution at coding sequence (CDS) and intron regions that is specific to these cells. A total of 1708 differentially expressed genes were identified after PARP1 knockdown using RNA-seq. Furthermore, transcriptome analysis also showed that selective variable splicing was globally regulated by PARP1 in HK-2 cells. By comparison of PARP1 RNA-seq and iRIP-seq data, we found 68 overlapping genes that are enriched in 'extracellular matrix' pathway. Follow-up identification of their interactions may contribute vital insights into the regulatory role of PARP1 as an RNA-binding protein in HK-2 cells.

Chromosome-length genome assembly and structural variations of the primal Basenji dog (Canis lupus familiaris) genome

BackgroundBasenjis are considered an ancient dog breed of central African origins that still live and hunt with tribesmen in the African Congo. Nicknamed the barkless dog, Basenjis possess unique phylogeny, geographical origins and traits, making their genome structure of great interest. The increasing number of available canid reference genomes allows us to examine the impact the choice of reference genome makes with regard to reference genome quality and breed relatedness.ResultsHere, we report two high quality de novo Basenji genome assemblies: a female, China (CanFam_Bas), and a male, Wags. We conduct pairwise comparisons and report structural variations between assembled genomes of three dog breeds: Basenji (CanFam_Bas), Boxer (CanFam3.1) and German Shepherd Dog (GSD) (CanFam_GSD). CanFam_Bas is superior to CanFam3.1 in terms of genome contiguity and comparable overall to the high quality CanFam_GSD assembly. By aligning short read data from 58 representative dog breeds to three reference genomes, we demonstrate how the choice of reference genome significantly impacts both read mapping and variant detection.ConclusionsThe growing number of high-quality canid reference genomes means the choice of reference genome is an increasingly critical decision in subsequent canid variant analyses. The basal position of the Basenji makes it suitable for variant analysis for targeted applications of specific dog breeds. However, we believe more comprehensive analyses across the entire family of canids is more suited to a pangenome approach. Collectively this work highlights the importance the choice of reference genome makes in all variation studies.

Comparative Genomics and Environmental Distribution of Large dsDNA Viruses in the Family Asfarviridae.

The family Asfarviridae is a group of nucleo-cytoplasmic large DNA viruses (NCLDVs) of which African swine fever virus (ASFV) is well-characterized. Recently the discovery of several Asfarviridae members other than ASFV has suggested that this family represents a diverse and cosmopolitan group of viruses, but the genomics and distribution of this family have not been studied in detail. To this end we analyzed five complete genomes and 35 metagenome-assembled genomes (MAGs) of viruses from this family to shed light on their evolutionary relationships and environmental distribution. The Asfarvirus MAGs derive from diverse marine, freshwater, and terrestrial habitats, underscoring the broad environmental distribution of this family. We present phylogenetic analyses using conserved marker genes and whole-genome comparison of pairwise average amino acid identity (AAI) values, revealing a high level of genomic divergence across disparate Asfarviruses. Further, we found that Asfarviridae genomes encode genes with diverse predicted metabolic roles and detectable sequence homology to proteins in bacteria, archaea, and eukaryotes, highlighting the genomic chimerism that is a salient feature of NCLDV. Our read mapping from Tara oceans metagenomic data also revealed that three Asfarviridae MAGs were present in multiple marine samples, indicating that they are widespread in the ocean. In one of these MAGs we identified four marker genes with > 95% AAI to genes sequenced from a virus that infects the dinoflagellate Heterocapsa circularisquama (HcDNAV). This suggests a potential host for this MAG, which would thereby represent a reference genome of a dinoflagellate-infecting giant virus. Together, these results show that Asfarviridae are ubiquitous, comprise similar sequence divergence as other NCLDV families, and include several members that are widespread in the ocean and potentially infect ecologically important protists.

Older orienteers perform better–is experience key?

With increasing age physical performance decreases. However, especially in orienteering, with the risk of running too fast in relation to map reading skills and thus of not being able to process important map information correctly a lower running speed can be an advantage. Further, with increasing age and the often-greater experience, the susceptibility to errors should decrease yielding to the aim of the study to analyze the relationship between average speed and age in senior orienteering. All national Competitions of Middle and Long Distance as well as the Swiss Championships in the Middle and Long Distance as well as in night orienteering were analyzed for the season 2018 and 2019 for all senior categories. Average running speed was calculated per category with the concept of performance km, whereby in 2018 ten competitions and in 2019 nine competitions yielding to a total sample size of 855 female and 950 male orienteers analyzed. Furthermore, polynomial analyses of second degree between average running speed and age were conducted with calculation of coefficient of determination and in addition multilinear regression analysis between average course time and age respectively distance absolved. Average age of peak performance was identified with 50.0 ± 2.3 years for men and 47.3 ± 3.1 for women in 2018 respectively 51.1 ± 3.1 years for men and 47.0 ± 3.0 for women in 2019. Furthermore, high coefficients of determination were identified with average values of 0.916 ± 0.061 in men and 0.910 ± 0.051 in women 2018 respectively 0.891 ± 0.102 in men and 0.913 ± 0.079 in women in 2019. The relatively high ages are probably due to the fact that Classic beginner mistakes such as running too fast compared to map reading skills become less common, which is reflected in an absolutely lower error rate and correspondingly leads to a less severe decrease in age-related performance. Avec l’âge, la performance physique diminue. Cependant, en course d’orientation une vitesse plus faible peut devenir un avantage, car courir trop vite peut exposer à analyser moins correctement les informations cartographiques. De plus, avec l’augmentation de l’âge l’expérience devient plus grande et la vulnérabilité aux erreurs devrait diminuer. L’objectif de cette étude était donc d’analyser la relation entre la vitesse moyenne et l’âge en course d’orientation senior. Toutes les compétitions nationales de moyenne et longue distance ainsi que les championnats suisses de moyenne et longue distance et les courses d’orientation de nuit ont été analysés pour les saisons 2018 et 2019 pour toutes les catégories seniors. La vitesse de course moyenne a été calculée par catégorie avec le concept de km de performance. En 2018 dix compétitions et en 2019 neuf compétitions donnant un échantillon total de 855 femmes et 950 hommes orienteurs analysés. De plus, des analyses polynomiales du second degré avec le coefficient de détermination entre la vitesse et l’âge ont été calculées et complétés par une analyse de régression multilinéaire entre le temps moyen des courses, et respectivement l’âge et la distance. L’âge moyen de la performance maximale était de 50,0 ± 2,3 ans pour les hommes et 47,3 ± 3,1 pour les femmes en 2018, et de 51,1 ± 3,1 ans pour les hommes et 47,0 ± 3,0 pour les femmes en 2019. Les coefficients de déterminations étaient élevés avec des valeurs moyennes de 0,916 ± 0,061 chez l’homme et 0,910 ± 0,051 chez la femme en 2018 et 0,891 ± 0,102 chez l’homme et 0,913 ± 0,079 chez la femme en 2019. Les âges relativement élevés sont expliqués par la moindre fréquence à ces âges des erreurs classiques que font les débutants, qui ont tendance à courir trop vite. De la sorte, la diminution de vitesse qui est observée avec l’âge n’a pas pour effet de diminuer la performance en course d’orientation.

A Comprehensive Map of mRNAs and Their Isoforms across All 14 Renal Tubule Segments of Mouse.

The repertoire of protein expression along the renal tubule depends both on regulation of transcription and regulation of alternative splicing that can generate multiple proteins from a single gene. A full-length, small-sample RNA-seq protocol profiled transcriptomes for all 14 renal tubule segments microdissected from mouse kidneys. This study identified >34,000 transcripts, including 3709 that were expressed in a segment-specific manner. All data are provided as an online resource (https://esbl.nhlbi.nih.gov/MRECA/Nephron/). Many of the genes expressed in unique patterns along the renal tubule were solute carriers, transcription factors, or G protein-coupled receptors that account for segment-specific function. Mapping the distribution of transcripts associated with Wnk-SPAK-PKA signaling, renin-angiotensin-aldosterone signaling, and cystic diseases of the kidney illustrated the applications of the online resource. The method allowed full-length mapping of RNA-seq reads, which facilitated comprehensive, unbiased characterization of alternative exon usage along the renal tubule, including known isoforms of Cldn10, Kcnj1 (ROMK), Slc12a1 (NKCC2), Wnk1, Stk39 (SPAK), and Slc14a2 (UT-A urea transporter). It also identified many novel isoforms with segment-specific distribution. These included variants associated with altered protein structure (Slc9a8, Khk, Tsc22d1, and Scoc), and variants that may affect untranslated, regulatory regions of transcripts (Pth1r, Pkar1a, and Dab2). Full-length, unbiased sequencing of transcripts identified gene-expression patterns along the mouse renal tubule. The data, provided as an online resource, include both quantitative and qualitative differences in transcripts. Identification of alternative splicing along the renal tubule may prove critical to understanding renal physiology and pathophysiology.

Higher Rates of Processed Pseudogene Acquisition in Humans and Three Great Apes Revealed by Long-Read Assemblies

LINE-1-mediated retrotransposition of protein-coding mRNAs is an active process in modern humans for both germline and somatic genomes. Prior works that surveyed human data mostly relied on detecting discordant mappings of paired-end short reads, or exon junctions contained in short reads. Moreover, there have been few genome-wide comparisons between gene retrocopies in great apes and humans. In this study, we introduced a more sensitive and accurate method to identify processed pseudogenes. Our method utilizes long-read assemblies, and more importantly, is able to provide full-length retrocopy sequences as well as flanking regions which are missed by short-read based methods. From 22 human individuals, we pinpointed 40 processed pseudogenes that are not present in the human reference genome GRCh38 and identified 17 pseudogenes that are in GRCh38 but absent from some input individuals. This represents a significantly higher discovery rate than previous reports (39 pseudogenes not in the reference genome out of 939 individuals). We also provided an overview of lineage-specific retrocopies in chimpanzee, gorilla, and orangutan genomes.

Validation strategy of a bioinformatics whole genome sequencing workflow for Shiga toxin-producing Escherichia coli using a reference collection extensively characterized with conventional methods.

Whole genome sequencing (WGS) enables complete characterization of bacterial pathogenic isolates at single nucleotide resolution, making it the ultimate tool for routine surveillance and outbreak investigation. The lack of standardization, and the variation regarding bioinformatics workflows and parameters, however, complicates interoperability among (inter)national laboratories. We present a validation strategy applied to a bioinformatics workflow for Illumina data that performs complete characterization of Shiga toxin-producing Escherichia coli (STEC) isolates including antimicrobial resistance prediction, virulence gene detection, serotype prediction, plasmid replicon detection and sequence typing. The workflow supports three commonly used bioinformatics approaches for the detection of genes and alleles: alignment with blast+, kmer-based read mapping with KMA, and direct read mapping with SRST2. A collection of 131 STEC isolates collected from food and human sources, extensively characterized with conventional molecular methods, was used as a validation dataset. Using a validation strategy specifically adopted to WGS, we demonstrated high performance with repeatability, reproducibility, accuracy, precision, sensitivity and specificity above 95 % for the majority of all assays. The WGS workflow is publicly available as a ‘push-button’ pipeline at https://galaxy.sciensano.be. Our validation strategy and accompanying reference dataset consisting of both conventional and WGS data can be used for characterizing the performance of various bioinformatics workflows and assays, facilitating interoperability between laboratories with different WGS and bioinformatics set-ups.

StLiter: A Novel Algorithm to Iteratively Build the Compacted de Bruijn Graph From Many Complete Genomes.

Recently, the compacted de Bruijn graph (cDBG) of complete genome sequences was successfully used in read mapping due to its ability to deal with the repetitions in genomes. However, current approaches are not flexible enough to fit frequently building the graphs with different k-mer lengths. Instead of building the graph directly, how can we build the compacted de Bruijin graph of longer k-mer based on the one of short k-mer? In this article, we present StLiter, a novel algorithm to build the compacted de Bruijn graph either directly from genome sequences or indirectly based on the graph of a short k-mer. For 100 simulated human genomes, StLiter can construct the graph of k-mer length 15-18 in 2.5-3.2 hours with maximal ∼70GB memory in the case of without considering the reverese complements of the reference genomes. And it costs 4.5-5.9 hours when considering the reverse complements. In experiments, we compared StLiter with TwoPaCo, the state-of-art method for building the graph, on 4 datasets. For k-mer length 15-18, StLiter can build the graph 5-9 times faster than TwoPaCo using less maximal memory cost. For k-mer length larger than 18, given the graph of a short (k- x)-mer, such as x= 1-2, compared with TwoPaCo building the graph directly, StLiter can also build the graph more efficiently. The source codes of StLiter can be downloaded from web site https://github.com/BioLab-cz/StLiter.