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September 22, 2019

MIRU-profiler: a rapid tool for determination of 24-loci MIRU-VNTR profiles from assembled genomes of Mycobacterium tuberculosis.

Tuberculosis (TB) resulted in an estimated 1.7 million deaths in the year 2016. The disease is caused by the members of Mycobacterium tuberculosis complex, which includes Mycobacterium tuberculosis, Mycobacterium bovis and other closely related TB causing organisms. In order to understand the epidemiological dynamics of TB, national TB control programs often conduct standardized genotyping at 24 Mycobacterial-Interspersed-Repetitive-Units (MIRU)-Variable-Number-of-Tandem-Repeats (VNTR) loci. With the advent of next generation sequencing technology, whole-genome sequencing (WGS) has been widely used for studying TB transmission. However, an open-source software that can connect WGS and MIRU-VNTR typing is currently unavailable, which hinders interlaboratory communication. In this manuscript, we introduce the MIRU-profiler program which could be used for prediction of MIRU-VNTR profile from WGS of M. tuberculosis.The MIRU-profiler is implemented in shell scripting language and depends on EMBOSS software. The in-silico workflow of MIRU-profiler is similar to those described in the laboratory manuals for genotyping M. tuberculosis. Given an input genome sequence, the MIRU-profiler computes alleles at the standard 24-loci based on in-silico PCR amplicon lengths. The final output is a tab-delimited text file detailing the 24-loci MIRU-VNTR pattern of the input sequence.The MIRU-profiler was validated on four datasets: complete genomes from NCBI-GenBank (n = 11), complete genomes for locally isolated strains sequenced using PacBio (n = 4), complete genomes for BCG vaccine strains (n = 2) and draft genomes based on 250 bp paired-end Illumina reads (n = 106).The digital MIRU-VNTR results were identical to the experimental genotyping results for complete genomes of locally isolated strains, BCG vaccine strains and five out of 11 genomes from the NCBI-GenBank. For draft genomes based on short Illumina reads, 21 out of 24 loci were inferred with a high accuracy, while a number of inaccuracies were recorded for three specific loci (ETRA, QUB11b and QUB26). One of the unique features of the MIRU-profiler was its ability to process multiple genomes in a batch. This feature was tested on all complete M. tuberculosis genome (n = 157), for which results were successfully obtained in approximately 14 min.The MIRU-profiler is a rapid tool for inference of digital MIRU-VNTR profile from the assembled genome sequences. The tool can accurately infer repeat numbers at the standard 24 or 21/24 MIRU-VNTR loci from the complete or draft genomes respectively. Thus, the tool is expected to bridge the communication gap between the laboratories using WGS and those using the conventional MIRU-VNTR typing.

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September 22, 2019

Evidence of non-tandemly repeated rDNAs and their intragenomic heterogeneity in Rhizophagus irregularis

Arbuscular mycorrhizal fungus (AMF) species are some of the most widespread symbionts of land plants. Our much improved reference genome assembly of a model AMF, Rhizophagus irregularis DAOM-181602 (total contigs?=?210), facilitated a discovery of repetitive elements with unusual characteristics. R. irregularis has only ten or 11 copies of complete 45S rDNAs, whereas the general eukaryotic genome has tens to thousands of rDNA copies. R. irregularis rDNAs are highly heterogeneous and lack a tandem repeat structure. These findings provide evidence for the hypothesis that rDNA heterogeneity depends on the lack of tandem repeat structures. RNA-Seq analysis confirmed that all rDNA variants are actively transcribed. Observed rDNA/rRNA polymorphisms may modulate translation by using different ribosomes depending on biotic and abiotic interactions. The non-tandem repeat structure and intragenomic heterogeneity of AMF rDNA/rRNA may facilitate successful adaptation to various environmental conditions, increasing host compatibility of these symbiotic fungi.

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September 22, 2019

Genotype-Corrector: improved genotype calls for genetic mapping in F2 and RIL populations.

F2 and recombinant inbred lines (RILs) populations are very commonly used in plant genetic mapping studies. Although genome-wide genetic markers like single nucleotide polymorphisms (SNPs) can be readily identified by a wide array of methods, accurate genotype calling remains challenging, especially for heterozygous loci and missing data due to low sequencing coverage per individual. Therefore, we developed Genotype-Corrector, a program that corrects genotype calls and imputes missing data to improve the accuracy of genetic mapping. Genotype-Corrector can be applied in a wide variety of genetic mapping studies that are based on low coverage whole genome sequencing (WGS) or Genotyping-by-Sequencing (GBS) related techniques. Our results show that Genotype-Corrector achieves high accuracy when applied to both synthetic and real genotype data. Compared with using raw or only imputed genotype calls, the linkage groups built by corrected genotype data show much less noise and significant distortions can be corrected. Additionally, Genotype-Corrector compares favorably to the popular imputation software LinkImpute and Beagle in both F2 and RIL populations. Genotype-Corrector is publicly available on GitHub at https://github.com/freemao/Genotype-Corrector .

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September 22, 2019

npInv: accurate detection and genotyping of inversions using long read sub-alignment.

Detection of genomic inversions remains challenging. Many existing methods primarily target inzversions with a non repetitive breakpoint, leaving inverted repeat (IR) mediated non-allelic homologous recombination (NAHR) inversions largely unexplored.We present npInv, a novel tool specifically for detecting and genotyping NAHR inversion using long read sub-alignment of long read sequencing data. We benchmark npInv with other tools in both simulation and real data. We use npInv to generate a whole-genome inversion map for NA12878 consisting of 30 NAHR inversions (of which 15 are novel), including all previously known NAHR mediated inversions in NA12878 with flanking IR less than 7kb. Our genotyping accuracy on this dataset was 94%. We used PCR to confirm the presence of two of these novel inversions. We show that there is a near linear relationship between the length of flanking IR and the minimum inversion size, without inverted repeats.The application of npInv shows high accuracy in both simulation and real data. The results give deeper insight into understanding inversion.

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September 22, 2019

Strand-seq enables reliable separation of long reads by chromosome via expectation maximization.

Current sequencing technologies are able to produce reads orders of magnitude longer than ever possible before. Such long reads have sparked a new interest in de novo genome assembly, which removes reference biases inherent to re-sequencing approaches and allows for a direct characterization of complex genomic variants. However, even with latest algorithmic advances, assembling a mammalian genome from long error-prone reads incurs a significant computational burden and does not preclude occasional misassemblies. Both problems could potentially be mitigated if assembly could commence for each chromosome separately.To address this, we show how single-cell template strand sequencing (Strand-seq) data can be leveraged for this purpose. We introduce a novel latent variable model and a corresponding Expectation Maximization algorithm, termed SaaRclust, and demonstrates its ability to reliably cluster long reads by chromosome. For each long read, this approach produces a posterior probability distribution over all chromosomes of origin and read directionalities. In this way, it allows to assess the amount of uncertainty inherent to sparse Strand-seq data on the level of individual reads. Among the reads that our algorithm confidently assigns to a chromosome, we observed more than 99% correct assignments on a subset of Pacific Bioscience reads with 30.1×?coverage. To our knowledge, SaaRclust is the first approach for the in silico separation of long reads by chromosome prior to assembly.https://github.com/daewoooo/SaaRclust.

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September 22, 2019

Genetic adaptation of a mevalonate pathway deficient mutant in Staphylococcus aureus.

In this study we addressed the question how a mevalonate (MVA)-auxotrophic Staphylococcus aureus?mvaS mutant can revert to prototrophy. This mutant couldn’t grow in the absence of MVA. However, after a long lag-phase of 4-6 days the mutant adapted from auxotrophic to prototrophic phenotype. During that time, it acquired two point mutations: One mutation in the coding region of the regulator gene spx, which resulted in an amino acid exchange that decreased Spx function. The other mutation in the upstream-element within the core-promoter of the mevalonolactone lactonase gene drp35. This mutation led to an increased expression of drp35. In repeated experiments the mutations always occurred in spx and drp35 and in the same order. The first detectable mutation appeared in spx and allowed slight growth; the second mutation, in drp35, increased growth further. Phenotypical characterizations of the mutant showed that small amounts of the lipid-carrier undecaprenol are synthesized, despite the lack of mvaS. The growth of the adapted clone, ?mvaSad, indicates that the mutations reawake a rescue bypass. We think that this bypass enters the MVA pathway at the stage of MVA, because blocking the pathway downstream of MVA led to growth arrest of the mutant. In addition, the lactonase Drp35 is able to convert mevalonolactone to MVA. Summarized, we describe here a mutation-based two-step adaptation process that allows resuscitation of growth of the ?mvaS mutant.

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September 22, 2019

The genome assembly of the fungal pathogen Pyrenochaeta lycopersici from Single-Molecule Real-Time sequencing sheds new light on its biological complexity.

The first draft genome sequencing of the non-model fungal pathogen Pyrenochaeta lycopersici showed an expansion of gene families associated with heterokaryon incompatibility and lacking of mating-type genes, providing insights into the genetic basis of this “imperfect” fungus which lost the ability to produce the sexual stage. However, due to the Illumina short-read technology, the draft genome was too fragmented to allow a comprehensive characterization of the genome, especially of the repetitive sequence fraction. In this work, the sequencing of another P. lycopersici isolate using long-read Single Molecule Real-Time sequencing technology was performed with the aim of obtaining a gapless genome. Indeed, a gapless genome assembly of 62.7 Mb was obtained, with a fraction of repetitive sequences representing 30% of the total bases. The gene content of the two P. lycopersici isolates was very similar, and the large difference in genome size (about 8 Mb) might be attributable to the high fraction of repetitive sequences detected for the new sequenced isolate. The role of repetitive elements, including transposable elements, in modulating virulence effectors is well established in fungal plant pathogens. Moreover, transposable elements are of fundamental importance in creating and re-modelling genes, especially in imperfect fungi. Their abundance in P. lycopersici, together with the large expansion of heterokaryon incompatibility genes in both sequenced isolates, suggest the presence of possible mechanisms alternative to gene re-assorting mediated by sexual recombination. A quite large fraction (~9%) of repetitive elements in P. lycopersici, has no homology with known classes, strengthening this hypothesis. The availability of a gapless genome of P. lycopersici allowed the in-depth analysis of its genome content, by annotating functional genes and TEs. This goal will be an important resource for shedding light on the evolution of the reproductive and pathogenic behaviour of this soilborne pathogen and the onset of a possible speciation within this species.

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September 22, 2019

Genome survey of the freshwater mussel Venustaconcha ellipsiformis (Bivalvia: Unionida) using a hybrid de novo assembly approach.

Freshwater mussels (Bivalvia: Unionida) serve an important role as aquatic ecosystem engineers but are one of the most critically imperilled groups of animals. Here, we used a combination of sequencing strategies to assemble and annotate a draft genome of Venustaconcha ellipsiformis, which will serve as a valuable genomic resource given the ecological value and unique “doubly uniparental inheritance” mode of mitochondrial DNA transmission of freshwater mussels. The genome described here was obtained by combining high-coverage short reads (65× genome coverage of Illumina paired-end and 11× genome coverage of mate-pairs sequences) with low-coverage Pacific Biosciences long reads (0.3× genome coverage). Briefly, the final scaffold assembly accounted for a total size of 1.54?Gb (366,926 scaffolds, N50?=?6.5 kb, with 2.3% of “N” nucleotides), representing 86% of the predicted genome size of 1.80?Gb, while over one third of the genome (37.5%) consisted of repeated elements and >85% of the core eukaryotic genes were recovered. Given the repeated genetic bottlenecks of V. ellipsiformis populations as a result of glaciations events, heterozygosity was also found to be remarkably low (0.6%), in contrast to most other sequenced bivalve species. Finally, we reassembled the full mitochondrial genome and found six polymorphic sites with respect to the previously published reference. This resource opens the way to comparative genomics studies to identify genes related to the unique adaptations of freshwater mussels and their distinctive mitochondrial inheritance mechanism.

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September 22, 2019

Genome-based population structure analysis of the strawberry plant pathogen Xanthomonas fragariae reveals two distinct groups that evolved independently before its species description.

Xanthomonas fragariae is a quarantine organism in Europe, causing angular leaf spots on strawberry plants. It is spreading worldwide in strawberry-producing regions due to import of plant material through trade and human activities. In order to resolve the population structure at the strain level, we have employed high-resolution molecular typing tools on a comprehensive strain collection representing global and temporal distribution of the pathogen. Clustered regularly interspaced short palindromic repeat regions (CRISPRs) and variable number of tandem repeats (VNTRs) were identified within the reference genome of X. fragariae LMG 25863 as a potential source of variation. Strains from our collection were whole-genome sequenced and used in order to identify variable spacers and repeats for discriminative purpose. CRISPR spacer analysis and multiple-locus VNTR analysis (MLVA) displayed a congruent population structure, in which two major groups and a total of four subgroups were revealed. The two main groups were genetically separated before the first X. fragariae isolate was described and are potentially responsible for the worldwide expansion of the bacterial disease. Three primer sets were designed for discriminating CRISPR-associated markers in order to streamline group determination of novel isolates. Overall, this study describes typing methods to discriminate strains and monitor the pathogen population structure, more especially in the view of a new outbreak of the pathogen.

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September 22, 2019

PBHoover and CigarRoller: a method for confident haploid variant calling on Pacific Biosciences data and its application to heterogeneous population analysis

Motivation: Single Molecule Real-Time (SMRT) sequencing has important and underutilized advantages that amplification-based platforms lack. Lack of systematic error (e.g. GC-bias), complete de novo assembly (including large repetitive regions) without scaffolding, can be mentioned. SMRT sequencing, however suffers from high random error rate and low sequencing depth (older chemistries). Here, we introduce PBHoover, software that uses a heuristic calling algorithm in order to make base calls with high certainty in low coverage regions. This software is also capable of mixed population detection with high sensitivity. PBHoovertextquoterights CigarRoller attachment improves sequencing depth in low-coverage regions through CIGAR-string correction. Results: We tested both modules on 348 M.tuberculosis clinical isolates sequenced on C1 or C2 chemistries. On average, CigarRoller improved percentage of usable read count from 68.9% to 99.98% in C1 runs and from 50% to 99% in C2 runs. Using the greater depth provided by CigarRoller, PBHoover was able to make base and variant calls 99.95% concordant with Sanger calls (QV33). PBHoover also detected antibiotic-resistant subpopulations that went undetected by Sanger. Using C1 chemistry, subpopulations as small as 9% of the total colony can be detected by PBHoover. This provides the most sensitive amplification-free molecular method for heterogeneity analysis and is in line with phenotypic methodstextquoteright sensitivity. This sensitivity significantly improves with the greater depth and lower error rate of the newer chemistries. Availability and Implementation: Executables are freely available under GNU GPL v3+ at http://www.gitlab.com/LPCDRP/pbhoover and http://www.gitlab.com/LPCDRP/CigarRoller. PBHoover is also available on bioconda: https://anaconda.org/bioconda/pbhoover.

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September 22, 2019

Sequencing of Panax notoginseng genome reveals genes involved in disease resistance and ginsenoside biosynthesis

Background: Panax notoginseng is a traditional Chinese herb with high medicinal and economic value. There has been considerable research on the pharmacological activities of ginsenosides contained in Panax spp.; however, very little is known about the ginsenoside biosynthetic pathway. Results: We reported the first de novo genome of 2.36 Gb of sequences from P. notoginseng with 35,451 protein-encoding genes. Compared to other plants, we found notable gene family contraction of disease-resistance genes in P. notoginseng, but notable expansion for several ATP-binding cassette (ABC) transporter subfamilies, such as the Gpdr subfamily, indicating that ABCs might be an additional mechanism for the plant to cope with biotic stress. Combining eight transcriptomes of roots and aerial parts, we identified several key genes, their transcription factor binding sites and all their family members involved in the synthesis pathway of ginsenosides in P. notoginseng, including dammarenediol synthase, CYP716 and UGT71. Conclusions: The complete genome analysis of P. notoginseng, the first in genus Panax, will serve as an important reference sequence for improving breeding and cultivation of this important nutraceutical and medicinal but vulnerable plant species.

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September 22, 2019

The integrative conjugative element clc (ICEclc) of Pseudomonas aeruginosa JB2.

Integrative conjugative elements (ICE) are a diverse group of chromosomally integrated, self-transmissible mobile genetic elements (MGE) that are active in shaping the functions of bacteria and bacterial communities. Each type of ICE carries a characteristic set of core genes encoding functions essential for maintenance and self-transmission, and cargo genes that endow on hosts phenotypes beneficial for niche adaptation. An important area to which ICE can contribute beneficial functions is the biodegradation of xenobiotic compounds. In the biodegradation realm, the best-characterized ICE is ICEclc, which carries cargo genes encoding for ortho-cleavage of chlorocatechols (clc genes) and aminophenol metabolism (amn genes). The element was originally identified in the 3-chlorobenzoate-degrader Pseudomonas knackmussii B13, and the closest relative is a nearly identical element in Burkholderia xenovorans LB400 (designated ICEclc-B13 and ICEclc-LB400, respectively). In the present report, genome sequencing of the o-chlorobenzoate degrader Pseudomonas aeruginosa JB2 was used to identify a new member of the ICEclc family, ICEclc-JB2. The cargo of ICEclc-JB2 differs from that of ICEclc-B13 and ICEclc-LB400 in consisting of a unique combination of genes that encode for the utilization of o-halobenzoates and o-hydroxybenzoate as growth substrates (ohb genes and hyb genes, respectively) and which are duplicated in a tandem repeat. Also, ICEclc-JB2 lacks an operon of regulatory genes (tciR-marR-mfsR) that is present in the other two ICEclc, and which controls excision from the host. Thus, the mechanisms regulating intracellular behavior of ICEclc-JB2 may differ from that of its close relatives. The entire tandem repeat in ICEclc-JB2 can excise independently from the element in a process apparently involving transposases/insertion sequence associated with the repeats. Excision of the repeats removes important niche adaptation genes from ICEclc-JB2, rendering it less beneficial to the host. However, the reduced version of ICEclc-JB2 could now acquire new genes that might be beneficial to a future host and, consequently, to the survival of ICEclc-JB2. Collectively, the present identification and characterization of ICEclc-JB2 provides insights into roles of MGE in bacterial niche adaptation and the evolution of catabolic pathways for biodegradation of xenobiotic compounds.

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September 22, 2019

Comparative genomics and genotype-phenotype associations in Bifidobacterium breve.

Bifidobacteria are common members of the gastro-intestinal microbiota of a broad range of animal hosts. Their successful adaptation to this particular niche is linked to their saccharolytic metabolism, which is supported by a wide range of glycosyl hydrolases. In the current study a large-scale gene-trait matching (GTM) effort was performed to explore glycan degradation capabilities in B. breve. By correlating the presence/absence of genes and associated genomic clusters with growth/no-growth patterns across a dataset of 20 Bifidobacterium breve strains and nearly 80 different potential growth substrates, we not only validated the approach for a number of previously characterized carbohydrate utilization clusters, but we were also able to discover novel genetic clusters linked to the metabolism of salicin and sucrose. Using GTM, genetic associations were also established for antibiotic resistance and exopolysaccharide production, thereby identifying (novel) bifidobacterial antibiotic resistance markers and showing that the GTM approach is applicable to a variety of phenotypes. Overall, the GTM findings clearly expand our knowledge on members of the B. breve species, in particular how their variable genetic features can be linked to specific phenotypes.

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September 22, 2019

Complete genome sequence of Enterococcus durans KLDS6.0933, a potential probiotic strain with high cholesterol removal ability

Enterococci are commensal bacteria in the mammalian gastrointestinal tract which play an important role in the production of various fermented foods. Thus, certain enterococcal strains are commonly used as probiotics to confer health benefits to human and animals. Enterococcus durans KLDS6.0933 is a potential probiotic strain with high cholesterol removal ability, which was isolated from traditional naturally fermented cream in Inner Mongolia of China. To better understand the genetic basis of the probiotic properties of this strain, the whole-genome sequence was performed using the PacBio RSII platform.

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September 22, 2019

Integrating long-range connectivity information into de Bruijn graphs.

The de Bruijn graph is a simple and efficient data structure that is used in many areas of sequence analysis including genome assembly, read error correction and variant calling. The data structure has a single parameter k, is straightforward to implement and is tractable for large genomes with high sequencing depth. It also enables representation of multiple samples simultaneously to facilitate comparison. However, unlike the string graph, a de Bruijn graph does not retain long range information that is inherent in the read data. For this reason, applications that rely on de Bruijn graphs can produce sub-optimal results given their input data.We present a novel assembly graph data structure: the Linked de Bruijn Graph (LdBG). Constructed by adding annotations on top of a de Bruijn graph, it stores long range connectivity information through the graph. We show that with error-free data it is possible to losslessly store and recover sequence from a Linked de Bruijn graph. With assembly simulations we demonstrate that the LdBG data structure outperforms both our de Bruijn graph and the String Graph Assembler (SGA). Finally we apply the LdBG to Klebsiella pneumoniae short read data to make large (12 kbp) variant calls, which we validate using PacBio sequencing data, and to characterize the genomic context of drug-resistance genes.Linked de Bruijn Graphs and associated algorithms are implemented as part of McCortex, which is available under the MIT license at https://github.com/mcveanlab/mccortex.Supplementary data are available at Bioinformatics online.

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