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April 21, 2020

A survey and evaluations of histogram-based statistics in alignment-free sequence comparison.

Since the dawn of the bioinformatics field, sequence alignment scores have been the main method for comparing sequences. However, alignment algorithms are quadratic, requiring long execution time. As alternatives, scientists have developed tens of alignment-free statistics for measuring the similarity between two sequences.We surveyed tens of alignment-free k-mer statistics. Additionally, we evaluated 33 statistics and multiplicative combinations between the statistics and/or their squares. These statistics are calculated on two k-mer histograms representing two sequences. Our evaluations using global alignment scores revealed that the majority of the statistics are sensitive and capable of finding similar sequences to a query sequence. Therefore, any of these statistics can filter out dissimilar sequences quickly. Further, we observed that multiplicative combinations of the statistics are highly correlated with the identity score. Furthermore, combinations involving sequence length difference or Earth Mover’s distance, which takes the length difference into account, are always among the highest correlated paired statistics with identity scores. Similarly, paired statistics including length difference or Earth Mover’s distance are among the best performers in finding the K-closest sequences. Interestingly, similar performance can be obtained using histograms of shorter words, resulting in reducing the memory requirement and increasing the speed remarkably. Moreover, we found that simple single statistics are sufficient for processing next-generation sequencing reads and for applications relying on local alignment. Finally, we measured the time requirement of each statistic. The survey and the evaluations will help scientists with identifying efficient alternatives to the costly alignment algorithm, saving thousands of computational hours.The source code of the benchmarking tool is available as Supplementary Materials. © The Author 2017. Published by Oxford University Press.

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April 21, 2020

Blast Fungal Genomes Show Frequent Chromosomal Changes, Gene Gains and Losses, and Effector Gene Turnover.

Pyricularia is a fungal genus comprising several pathogenic species causing the blast disease in monocots. Pyricularia oryzae, the best-known species, infects rice, wheat, finger millet, and other crops. As past comparative and population genomics studies mainly focused on isolates of P. oryzae, the genomes of the other Pyricularia species have not been well explored. In this study, we obtained a chromosomal-level genome assembly of the finger millet isolate P. oryzae MZ5-1-6 and also highly contiguous assemblies of Pyricularia sp. LS, P. grisea, and P. pennisetigena. The differences in the genomic content of repetitive DNA sequences could largely explain the variation in genome size among these new genomes. Moreover, we found extensive gene gains and losses and structural changes among Pyricularia genomes, including a large interchromosomal translocation. We searched for homologs of known blast effectors across fungal taxa and found that most avirulence effectors are specific to Pyricularia, whereas many other effectors share homologs with distant fungal taxa. In particular, we discovered a novel effector family with metalloprotease activity, distinct from the well-known AVR-Pita family. We predicted 751 gene families containing putative effectors in 7 Pyricularia genomes and found that 60 of them showed differential expression in the P. oryzae MZ5-1-6 transcriptomes obtained under experimental conditions mimicking the pathogen infection process. In summary, this study increased our understanding of the structural, functional, and evolutionary genomics of the blast pathogen and identified new potential effector genes, providing useful data for developing crops with durable resistance. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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April 21, 2020

Chromosome-level genome assembly of Triplophysa tibetana, a fish adapted to the harsh high-altitude environment of the Tibetan Plateau.

Triplophysa is an endemic fish genus of the Tibetan Plateau in China. Triplophysa tibetana, which lives at a recorded altitude of ~4,000 m and plays an important role in the highland aquatic ecosystem, serves as an excellent model for investigating high-altitude environmental adaptation. However, evolutionary and conservation studies of T. tibetana have been limited by scarce genomic resources for the genus Triplophysa. In the present study, we applied PacBio sequencing and the Hi-C technique to assemble the T. tibetana genome. A 652-Mb genome with 1,325 contigs with an N50 length of 3.1 Mb was obtained. The 1,137 contigs were further assembled into 25 chromosomes, representing 98.7% and 80.47% of all contigs at the base and sequence number level, respectively. Approximately 260 Mb of sequence, accounting for ~39.8% of the genome, was identified as repetitive elements. DNA transposons (16.3%), long interspersed nuclear elements (12.4%) and long terminal repeats (11.0%) were the most repetitive types. In total, 24,372 protein-coding genes were predicted in the genome, and ~95% of the genes were functionally annotated via a search in public databases. Using whole genome sequence information, we found that T. tibetana diverged from its common ancestor with Danio rerio ~121.4 million years ago. The high-quality genome assembled in this work not only provides a valuable genomic resource for future population and conservation studies of T. tibetana, but it also lays a solid foundation for further investigation into the mechanisms of environmental adaptation of endemic fishes in the Tibetan Plateau. © 2019 John Wiley & Sons Ltd.

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April 21, 2020

Analysis of the Complete Genome Sequence of a Novel, Pseudorabies Virus Strain Isolated in Southeast Europe.

Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease giving rise to significant economic losses worldwide. Many countries have implemented national programs for the eradication of this virus. In this study, long-read sequencing was used to determine the nucleotide sequence of the genome of a novel PRV strain (PRV-MdBio) isolated in Serbia.In this study, a novel PRV strain was isolated and characterized. PRV-MdBio was found to exhibit similar growth properties to those of another wild-type PRV, the strain Kaplan. Single-molecule real-time (SMRT) sequencing has revealed that the new strain differs significantly in base composition even from strain Kaplan, to which it otherwise exhibits the highest similarity. We compared the genetic composition of PRV-MdBio to strain Kaplan and the China reference strain Ea and obtained that radical base replacements were the most common point mutations preceding conservative and silent mutations. We also found that the adaptation of PRV to cell culture does not lead to any tendentious genetic alteration in the viral genome.PRV-MdBio is a wild-type virus, which differs in base composition from other PRV strains to a relatively large extent.

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April 21, 2020

A global survey of full-length transcriptome of Ginkgo biloba reveals transcript variants involved in flavonoid biosynthesis

Ginkgo biloba, which contains flavonoids as bioactive components, is widely used in traditional Chinese medicine. Increasing the flavonoid production of medicinal plants through genetic engineering generally focuses on the key genes involved in flavonoid biosynthesis. However, the molecular mechanisms underlying such biosynthesis are not yet well understood. To understand these mechanisms, a combination of second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing was applied to G. biloba. Eight tissues were sampled for SMRT sequencing to generate a high-quality, full-length transcriptome database. From 23.36 Gb clean reads, 12,954 alternative polyadenylation events, 12,290 alternative splicing events, 929 fusion transcripts, 2,286 novel transcripts, and 1,270 lncRNAs were predicted by removing redundant reads. Further studies reveal that 7 AS, 5 lncRNA, and 6 fusion gene events were identified in flavonoid biosynthesis. A total of 12 gene modules were revealed to be involved in flavonoid metabolism structural genes and transcription factors by constructing co-expression networks. Weighted gene coexpression network analysis (WGCNA) analysis reveals that some hub genes operate during the biosynthesis by identifying transcription factors (TFs) and structure genes. Seven key hub genes were also identified by analyzing the correlation between gene expression level and flavonoids content. The results highlight the importance of SMRT sequencing of the full-length transcriptome in improving genome annotation and elucidating the gene regulation of flavonoid biosynthesis in G. biloba by providing a comprehensive set of reference transcripts.

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April 21, 2020

Transcriptome analysis reveals multiple signal network contributing to the Verticillium wilt resistance in eggplant

Verticillium wilt is a devastating disease in eggplants. In order to understand the molecular mechanism of disease resistance in eggplants, transcriptomes of Verticillium wilt infected eggplants were detected. A total of 480, 518, 887 and 1 046 Verticillium wilt related differentially expressed genes were identified at 6 (V6), 12 (V12), 24 (V24) and 48?h (V48), respectively. COG function classification revealed that most of DEGs functioned in “Amino acid transport and metabolism”, “Cytoskeleton” and “Cell motility”. In addition, compared the control plants (V0) to infected eggplants (V6-V48), a total of 111 common DEGs were identified. Except for “General function prediction only”, most of the DEGs enriched in “Signal transduction”. DEGs associated to different hormone signals, including GID1B, ROPGAP1, OPT3 and CDPK, were identified throughout the whole infection process. Cross-talk among defense signal pathways plays major roles in the Verticillium wilt disease resistance in eggplants.

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April 21, 2020

5’UTR-mediated regulation of Ataxin-1 expression.

Expression of mutant Ataxin-1 with an abnormally expanded polyglutamine domain is necessary for the onset and progression of spinocerebellar ataxia type 1 (SCA1). Understanding how Ataxin-1 expression is regulated in the human brain could inspire novel molecular therapies for this fatal, dominantly inherited neurodegenerative disease. Previous studies have shown that the ATXN1 3’UTR plays a key role in regulating the Ataxin-1 cellular pool via diverse post-transcriptional mechanisms. Here we show that elements within the ATXN1 5’UTR also participate in the regulation of Ataxin-1 expression. PCR and PacBio sequencing analysis of cDNA obtained from control and SCA1 human brain samples revealed the presence of three major, alternatively spliced ATXN1 5’UTR variants. In cell-based assays, fusion of these variants upstream of an EGFP reporter construct revealed significant and differential impacts on total EGFP protein output, uncovering a type of genetic rheostat-like function of the ATXN1 5’UTR. We identified ribosomal scanning of upstream AUG codons and increased transcript instability as potential mechanisms of regulation. Importantly, transcript-based analyses revealed significant differences in the expression pattern of ATXN1 5’UTR variants between control and SCA1 cerebellum. Together, the data presented here shed light into a previously unknown role for the ATXN1 5’UTR in the regulation of Ataxin-1 and provide new opportunities for the development of SCA1 therapeutics. Copyright © 2019. Published by Elsevier Inc.

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April 21, 2020

SMRT sequencing reveals differential patterns of methylation in two O111:H- STEC isolates from a hemolytic uremic syndrome outbreak in Australia.

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5′-CTGCm6AG-3′) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

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April 21, 2020

Insight into the microbial world of Bemisia tabaci cryptic species complex and its relationships with its host.

The 37 currently recognized Bemisia tabaci cryptic species are economically important species and contain both primary and secondary endosymbionts, but their diversity has never been mapped systematically across the group. To achieve this, PacBio sequencing of full-length bacterial 16S rRNA gene amplicons was carried out on 21 globally collected species in the B. tabaci complex, and two samples from B. afer were used here as outgroups. The microbial diversity was first explored across the major lineages of the whole group and 15 new putative bacterial sequences were observed. Extensive comparison of our results with previous endosymbiont diversity surveys which used PCR or multiplex 454 pyrosequencing platforms showed that the bacterial diversity was underestimated. To validate these new putative bacteria, one of them (Halomonas) was first confirmed to be present in MED B. tabaci using Hiseq2500 and FISH technologies. These results confirmed PacBio is a reliable and informative venue to reveal the bacterial diversity of insects. In addition, many new secondary endosymbiotic strains of Rickettsia and Arsenophonus were found, increasing the known diversity in these groups. For the previously described primary endosymbionts, one Portiera Operational Taxonomic Units (OTU) was shared by all B. tabaci species. The congruence of the B. tabaci-host and Portiera phylogenetic trees provides strong support for the hypothesis that primary endosymbionts co-speciated with their hosts. Likewise, a comparison of bacterial alpha diversities, Principal Coordinate Analysis, indistinct endosymbiotic communities harbored by different species and the co-divergence analyses suggest a lack of association between overall microbial diversity with cryptic species, further indicate that the secondary endosymbiont-mediated speciation is unlikely to have occurred in the B. tabaci species group.

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April 21, 2020

Large Enriched Fragment Targeted Sequencing (LEFT-SEQ) Applied to Capture of Wolbachia Genomes.

Symbiosis is a major force of evolutionary change, influencing virtually all aspects of biology, from population ecology and evolution to genomics and molecular/biochemical mechanisms of development and reproduction. A remarkable example is Wolbachia endobacteria, present in some parasitic nematodes and many arthropod species. Acquisition of genomic data from diverse Wolbachia clades will aid in the elucidation of the different symbiotic mechanisms(s). However, challenges of de novo assembly of Wolbachia genomes include the presence in the sample of host DNA: nematode/vertebrate or insect. We designed biotinylated probes to capture large fragments of Wolbachia DNA for sequencing using PacBio technology (LEFT-SEQ: Large Enriched Fragment Targeted Sequencing). LEFT-SEQ was used to capture and sequence four Wolbachia genomes: the filarial nematode Brugia malayi, wBm, (21-fold enrichment), Drosophila mauritiana flies (2 isolates), wMau (11-fold enrichment), and Aedes albopictus mosquitoes, wAlbB (200-fold enrichment). LEFT-SEQ resulted in complete genomes for wBm and for wMau. For wBm, 18 single-nucleotide polymorphisms (SNPs), relative to the wBm reference, were identified and confirmed by PCR. A limit of LEFT-SEQ is illustrated by the wAlbB genome, characterized by a very high level of insertion sequences elements (ISs) and DNA repeats, for which only a 20-contig draft assembly was achieved.

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April 21, 2020

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation.

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum ß-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.

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April 21, 2020

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.

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April 21, 2020

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions. A significant fraction of changes in gene expression can be explained by positive selection on single mutations that either create or disrupt transcriptional start sites (TSS). Furthermore, we show that clinical strains have different methyltransferases inactivated and thus different methylation patterns. Under the tested conditions, differential methylation has a minor direct role on transcriptomic differences between strains. However, disruption of a methyltransferase in one clinical strain revealed important expression differences suggesting indirect mechanisms of expression regulation. Our study demonstrates that variation in transcriptional profiles are mainly due to TSS mutations and have likely evolved due to differences in host characteristics.

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April 21, 2020

CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.

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