Whole Genome Shotgun Reads Research Articles

Development of polymorphic markers in the immune gene complex loci of cattle

The addition of cattle health and immunity traits to genomic selection indices holds promise to increase individual animal longevity and productivity, and decrease economic losses from disease. However, highly variable genomic loci that contain multiple immune-related genes were poorly assembled in the first iterations of the cattle reference genome assembly and underrepresented during the development of most commercial genotyping platforms. As a consequence, there is a paucity of genetic markers within these loci that may track haplotypes related to disease susceptibility. By using hierarchical assembly of bacterial artificial chromosome inserts spanning 3 of these immune-related gene regions, we were able to assemble multiple full-length haplotypes of the major histocompatibility complex, the leukocyte receptor complex, and the natural killer cell complex. Using these new assemblies and the recently released ARS-UCD1.2 reference, we aligned whole-genome shotgun reads from 125 sequenced Holstein bulls to discover candidate variants for genetic marker development. We selected 124 SNPs, using heuristic and statistical models to develop a custom genotyping panel. In a proof-of-principle study, we used this custom panel to genotype 1,797 Holstein cows exposed to bovine tuberculosis (bTB) that were the subject of a previous GWAS study using the Illumina BovineHD array. Although we did not identify any significant association of bTB phenotypes with these new genetic markers, 2 markers exhibited substantial effects on bTB phenotypic prediction. The models and parameters trained in this study serve as a guide for future marker discovery surveys particularly in previously unassembled regions of the cattle genome.

Visualization-assisted binning of metagenome assemblies reveals potential new pathogenic profiles in idiopathic travelers\u2019 diarrhea

BackgroundTravelers’ diarrhea (TD) is often caused by enterotoxigenic Escherichia coli, enteroaggregative E. coli, other bacterial pathogens, Norovirus, and occasionally parasites. Nevertheless, standard diagnostic methods fail to identify pathogens in more than 40% of TD patients. It is predicted that new pathogens may be causative agents of the disease.ResultsWe performed a comprehensive amplicon and whole genome shotgun (WGS) metagenomic study of the fecal microbiomes from 23 TD patients and seven healthy travelers, all of which were negative for the known etiologic agents of TD based on standard microbiological and immunological assays. Abnormal and diverse taxonomic profiles in TD samples were revealed. WGS reads were assembled and the resulting contigs were visualized using multiple query types. A semi-manual workflow was applied to isolate independent genomes from metagenomic pools. A total of 565 genome bins were extracted, 320 of which were complete enough to be characterized as cellular genomes; 160 were viral genomes. We made predictions of the etiology of disease for many of the individual subjects based on the properties and features of the recovered genomes. Multiple patients with low-diversity metagenomes were predominated by one to several E. coli strains. Functional annotation allowed prediction of pathogenic type in many cases. Five patients were co-infected with E. coli and other members of Enterobacteriaceae, including Enterobacter, Klebsiella, and Citrobacter; these may represent blooms of organisms that appear following secretory diarrhea. New “dark matter” microbes were observed in multiple samples. In one, we identified a novel TM7 genome that phylogenetically clustered with a sludge isolate; it carries genes encoding potential virulence factors. In multiple samples, we observed high proportions of putative novel viral genomes, some of which form clusters with the ubiquitous gut virus, crAssphage. The total relative abundance of viruses was significantly higher in healthy travelers versus TD patients.ConclusionOur study highlights the strength of assembly-based metagenomics, especially the manually curated, visualization-assisted binning of contigs, in resolving unusual and under-characterized pathogenic profiles of human-associated microbiomes. Results show that TD may be polymicrobial, with multiple novel cellular and viral strains as potential players in the diarrheal disease.

Karyotypes and Distribution of Tandem Repeat Sequences in Brassica nigra Determined by Fluorescence in situ Hybridization

Whole-genome shotgun reads were analyzed to determine the repeat sequence composition in the genome of black mustard, Brassica nigra (L.) Koch. The analysis showed that satellite DNA sequences are very abundant in the black mustard genome. The distribution pattern of 7 new tandem repeats (BnSAT13, BnSAT28, BnSAT68, BnSAT76, BnSAT114, BnSAT180, and BnSAT200) on black mustard chromosomes was visualized using fluorescence in situ hybridization (FISH). The FISH signals of BnSAT13 and BnSAT76 provided useful cytogenetic markers; their position and fluorescence intensity allowed for unambiguous identification of all 8 somatic metaphase chromosomes. A karyotype showing the location and fluorescence intensity of these tandem repeat sequences together with the position of rDNAs and centromeric retrotransposons of Brassica (CRB) was constructed. The establishment of the FISH-based karyotype in B. nigra provides valuable information that can be used in detailed analyses of B. nigra accessions and derived allopolyploid Brassica species containing the B genome.

De Novo Assembly of Complete Chloroplast Genomes from Non-model Species Based on a K-mer Frequency-Based Selection of Chloroplast Reads from Total DNA Sequences.

Whole Genome Shotgun (WGS) sequences of plant species often contain an abundance of reads that are derived from the chloroplast genome. Up to now these reads have generally been identified and assembled into chloroplast genomes based on homology to chloroplasts from related species. This re-sequencing approach may select against structural differences between the genomes especially in non-model species for which no close relatives have been sequenced before. The alternative approach is to de novo assemble the chloroplast genome from total genomic DNA sequences. In this study, we used k-mer frequency tables to identify and extract the chloroplast reads from the WGS reads and assemble these using a highly integrated and automated custom pipeline. Our strategy includes steps aimed at optimizing assemblies and filling gaps which are left due to coverage variation in the WGS dataset. We have successfully de novo assembled three complete chloroplast genomes from plant species with a range of nuclear genome sizes to demonstrate the universality of our approach: Solanum lycopersicum (0.9 Gb), Aegilops tauschii (4 Gb) and Paphiopedilum henryanum (25 Gb). We also highlight the need to optimize the choice of k and the amount of data used. This new and cost-effective method for de novo short read assembly will facilitate the study of complete chloroplast genomes with more accurate analyses and inferences, especially in non-model plant genomes.

KAT: a K-mer analysis toolkit to quality control NGS datasets and genome assemblies.

Motivation De novo assembly of whole genome shotgun (WGS) next-generation sequencing (NGS) data benefits from high-quality input with high coverage. However, in practice, determining the quality and quantity of useful reads quickly and in a reference-free manner is not trivial. Gaining a better understanding of the WGS data, and how that data is utilized by assemblers, provides useful insights that can inform the assembly process and result in better assemblies.ResultsWe present the K-mer Analysis Toolkit (KAT): a multi-purpose software toolkit for reference-free quality control (QC) of WGS reads and de novo genome assemblies, primarily via their k-mer frequencies and GC composition. KAT enables users to assess levels of errors, bias and contamination at various stages of the assembly process. In this paper we highlight KAT’s ability to provide valuable insights into assembly composition and quality of genome assemblies through pairwise comparison of k-mers present in both input reads and the assemblies.Availability and ImplementationKAT is available under the GPLv3 license at: https://github.com/TGAC/KAT.Supplementary information Supplementary data are available at Bioinformatics online.

Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement.

Upland cotton is a model for polyploid crop domestication and transgenic improvement. Here we sequenced the allotetraploid Gossypium hirsutum L. acc. TM-1 genome by integrating whole-genome shotgun reads, bacterial artificial chromosome (BAC)-end sequences and genotype-by-sequencing genetic maps. We assembled and annotated 32,032 A-subgenome genes and 34,402 D-subgenome genes. Structural rearrangements, gene loss, disrupted genes and sequence divergence were more common in the A subgenome than in the D subgenome, suggesting asymmetric evolution. However, no genome-wide expression dominance was found between the subgenomes. Genomic signatures of selection and domestication are associated with positively selected genes (PSGs) for fiber improvement in the A subgenome and for stress tolerance in the D subgenome. This draft genome sequence provides a resource for engineering superior cotton lines.

LTR retrotransposon dynamics in the evolution of the olive (Olea europaea) genome.

Improved knowledge of genome composition, especially of its repetitive component, generates important information for both theoretical and applied research. The olive repetitive component is made up of two main classes of sequences: tandem repeats and retrotransposons (REs). In this study, we provide characterization of a sample of 254 unique full-length long terminal repeat (LTR) REs. In the sample, Ty1-Copia elements were more numerous than Ty3-Gypsy elements. Mapping a large set of Illumina whole-genome shotgun reads onto the identified retroelement set revealed that Gypsy elements are more redundant than Copia elements. The insertion time of intact retroelements was estimated based on sister LTR’s divergence. Although some elements inserted relatively recently, the mean insertion age of the isolated retroelements is around 18 million yrs. Gypsy and Copia retroelements showed different waves of transposition, with Gypsy elements especially active between 10 and 25 million yrs ago and nearly inactive in the last 7 million yrs. The occurrence of numerous solo-LTRs related to isolated full-length retroelements was ascertained for two Gypsy elements and one Copia element. Overall, the results reported in this study show that RE activity (both retrotransposition and DNA loss) has impacted the olive genome structure in more ancient times than in other angiosperms.

RepARK—de novo creation of repeat libraries from whole-genome NGS reads

Generation of repeat libraries is a critical step for analysis of complex genomes. In the era of next-generation sequencing (NGS), such libraries are usually produced using a whole-genome shotgun (WGS) derived reference sequence whose completeness greatly influences the quality of derived repeat libraries. We describe here a de novo repeat assembly method—RepARK (Repetitive motif detection by Assembly of Repetitive K-mers)—which avoids potential biases by using abundant k-mers of NGS WGS reads without requiring a reference genome. For validation, repeat consensuses derived from simulated and real Drosophila melanogaster NGS WGS reads were compared to repeat libraries generated by four established methods. RepARK is orders of magnitude faster than the other methods and generates libraries that are: (i) composed almost entirely of repetitive motifs, (ii) more comprehensive and (iii) almost completely annotated by TEclass. Additionally, we show that the RepARK method is applicable to complex genomes like human and can even serve as a diagnostic tool to identify repetitive sequences contaminating NGS datasets.

Major repeat components covering one‐third of the ginseng (Panax ginseng C.A. Meyer) genome and evidence for allotetraploidy

Ginseng (Panax ginseng) is a famous medicinal herb, but the composition and structure of its genome are largely unknown. Here we characterized the major repeat components and inspected their distribution in the ginseng genome. By analyzing three repeat-rich bacterial artificial chromosome (BAC) sequences from ginseng, we identified complex insertion patterns of 34 long terminal repeat retrotransposons (LTR-RTs) and 11 LTR-RT derivatives accounting for more than 80% of the BAC sequences. The LTR-RTs were classified into three Ty3/gypsy (PgDel, PgTat and PgAthila) and two Ty1/Copia (PgTork and PgOryco) families. Mapping of 30-Gbp Illumina whole-genome shotgun reads to the BAC sequences revealed that these five LTR-RT families occupy at least 34% of the ginseng genome. The Ty3/Gypsy families were predominant, comprising 74 and 33% of the BAC sequences and the genome, respectively. In particular, the PgDel family accounted for 29% of the genome and presumably played major roles in enlargement of the size of the ginseng genome. Fluorescence in situ hybridization (FISH) revealed that the PgDel1 elements are distributed throughout the chromosomes along dispersed heterochromatic regions except for ribosomal DNA blocks. The intensity of the PgDel2 FISH signals was biased toward 24 out of 48 chromosomes. Unique gene probes showed two pairs of signals with different locations, one pair in subtelomeric regions on PgDel2-rich chromosomes and the other in interstitial regions on PgDel2-poor chromosomes, demonstrating allotetraploidy in ginseng. Our findings promote understanding of the evolution of the ginseng genome and of that of related species in the Araliaceae.

Centromere reference models for human chromosomes X and Y satellite arrays

The human genome sequence remains incomplete, with multimegabase-sized gaps representing the endogenous centromeres and other heterochromatic regions. Available sequence-based studies within these sites in the genome have demonstrated a role in centromere function and chromosome pairing, necessary to ensure proper chromosome segregation during cell division. A common genomic feature of these regions is the enrichment of long arrays of near-identical tandem repeats, known as satellite DNAs, which offer a limited number of variant sites to differentiate individual repeat copies across millions of bases. This substantial sequence homogeneity challenges available assembly strategies and, as a result, centromeric regions are omitted from ongoing genomic studies. To address this problem, we utilize monomer sequence and ordering information obtained from whole-genome shotgun reads to model two haploid human satellite arrays on chromosomes X and Y, resulting in an initial characterization of 3.83 Mb of centromeric DNA within an individual genome. To further expand the utility of each centromeric reference sequence model, we evaluate sites within the arrays for short-read mappability and chromosome specificity. Because satellite DNAs evolve in a concerted manner, we use these centromeric assemblies to assess the extent of sequence variation among 366 individuals from distinct human populations. We thus identify two satellite array variants in both X and Y centromeres, as determined by array length and sequence composition. This study provides an initial sequence characterization of a regional centromere and establishes a foundation to extend genomic characterization to these sites as well as to other repeat-rich regions within complex genomes.

Molecular Identification of Carnosine Synthase as ATP-grasp Domain-containing Protein 1 (ATPGD1)

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyryl-L-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 and 90 kDa were present in the preparation. Mass spectrometry analysis of these polypeptides did not yield any meaningful candidate. Carnosine formation catalyzed by the purified enzyme was accompanied by a stoichiometric formation, not of AMP, but of ADP, suggesting that carnosine synthase belongs to the "ATP-grasp family" of ligases. A data base mining approach identified ATPGD1 as a likely candidate. As this protein was absent from chicken protein data bases, we reconstituted its sequence from a PCR-amplified cDNA and found it to fit with the 100-kDa polypeptide of the chicken carnosine synthase preparation. Mouse and human ATPGD1 were expressed in HEK293T cells, purified to homogeneity, and shown to catalyze the formation of carnosine, as confirmed by mass spectrometry, and of homocarnosine. Specificity studies carried out on all three enzymes were in agreement with published data. In particular, they acted with 15-25-fold higher catalytic efficiencies on beta-alanine than on gamma-aminobutyrate. The identification of the gene encoding carnosine synthase will help for a better understanding of the biological functions of carnosine and related dipeptides, which still remain largely unknown.

Bos taurus genome assembly

BackgroundWe present here the assembly of the bovine genome. The assembly method combines the BAC plus WGS local assembly used for the rat and sea urchin with the whole genome shotgun (WGS) only assembly used for many other animal genomes including the rhesus macaque.ResultsThe assembly process consisted of multiple phases: First, BACs were assembled with BAC generated sequence, then subsequently in combination with the individual overlapping WGS reads. Different assembly parameters were tested to separately optimize the performance for each BAC assembly of the BAC and WGS reads. In parallel, a second assembly was produced using only the WGS sequences and a global whole genome assembly method. The two assemblies were combined to create a more complete genome representation that retained the high quality BAC-based local assembly information, but with gaps between BACs filled in with the WGS-only assembly. Finally, the entire assembly was placed on chromosomes using the available map information.Over 90% of the assembly is now placed on chromosomes. The estimated genome size is 2.87 Gb which represents a high degree of completeness, with 95% of the available EST sequences found in assembled contigs. The quality of the assembly was evaluated by comparison to 73 finished BACs, where the draft assembly covers between 92.5 and 100% (average 98.5%) of the finished BACs. The assembly contigs and scaffolds align linearly to the finished BACs, suggesting that misassemblies are rare. Genotyping and genetic mapping of 17,482 SNPs revealed that more than 99.2% were correctly positioned within the Btau_4.0 assembly, confirming the accuracy of the assembly.ConclusionThe biological analysis of this bovine genome assembly is being published, and the sequence data is available to support future bovine research.

Assisted assembly: how to improve a de novo genome assembly by using related species

A method is described for improving low sequence coverage genome assemblies

Distribution of the tandem repeat sequences and karyotyping in cucumber (Cucumis sativus L.) by fluorescence in situ hybridization

We analyzed repeat sequences composition in the genome of cucumber inbred line 9930 using whole-genome shotgun reads. The analysis showed that satellite DNA sequences are the most dominant components in the cucumber genome. The distribution pattern of several tandem repeat sequences (Type I/II, Type III and Type IV) on cucumber chromosomes was visualized using fluorescence in situ hybridization (FISH). The FISH signals of the Type III and 45S rDNA provide useful cytogenetic markers, whose position and fluorescence intensity allow for easy identification of all somatic metaphase chromosomes. A karyotype showing the position and fluorescence intensity of several tandem repeat sequences is constructed. The establishment of this FISH-based karyotype has created the basis for the integration of molecular, genetic and cytogenetic maps in Cucumis sativus and for the ultimate genome sequencing project as well.

Beware of mis-assembled genomes

Beware of mis-assembled genomes

Leveraging the mouse genome for gene prediction in human: from whole-genome shotgun reads to a global synteny map.

The availability of draft sequences for both the mouse and human genomes makes it possible, for the first time, to annotate whole mammalian genomes using comparative methods. TWINSCAN is a gene-prediction system that combines the methods of single-genome predictors like GENSCAN with information derived from genome comparison, thereby improving accuracy. Because TWINSCAN uses genomic sequence only, it is less biased toward highly and/or ubiquitously expressed genes than GENEWISE, GENOMESCAN, and other methods based on evidence derived from transcripts. We show that TWINSCAN improves gene prediction in human using intermediate products from various stages of the sequencing and analysis of the mouse genome, from low-redundancy, whole-genome shotgun reads to the draft assembly and the synteny map. TWINSCAN improves on the prior state of the art even when alignments from only 1X coverage of the mouse genome are available. Gene prediction accuracy improves steadily from 1X through 3X, more slowly from 3X to 4X, and relatively little thereafter. The assembly and the synteny map greatly speed the computations, however. Our human annotation using the mouse assembly is conservative, predicting only 25,622 genes, and appears to be one of the best de novo annotations of the human genome to date.