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Asset Tag: PacBio RS II

September 22, 2019

Detecting epigenetic motifs in low coverage and metagenomics settings.

It has recently become possible to rapidly and accurately detect epigenetic signatures in bacterial genomes using third generation sequencing data. Monitoring the speed at which a single polymerase inserts a base in the read strand enables one to infer whether a modification is present at that specific site on the template strand. These sites can be challenging to detect in the absence of high coverage and reliable reference genomes.Here we provide a new method for detecting epigenetic motifs in bacteria on datasets with low-coverage, with incomplete references, and with mixed samples (i.e. metagenomic data). Our approach treats motif inference as a kmer comparison problem. First, genomes (or contigs) are deconstructed into kmers. Then, native genome-wide distributions of interpulse durations (IPDs) for kmers are compared with corresponding whole genome amplified (WGA, modification free) IPD distributions using log likelihood ratios. Finally, kmers are ranked and greedily selected by iteratively correcting for sequences within a particular kmer’s neighborhood.Our method can detect multiple types of modifications, even at very low-coverage and in the presence of mixed genomes. Additionally, we are able to predict modified motifs when genomes with “neighbor” modified motifs exist within the sample. Lastly, we show that these motifs can provide an alternative source of information by which to cluster metagenomics contigs and that iterative refinement on these clustered contigs can further improve both sensitivity and specificity of motif detection.https://github.com/alibashir/EMMCKmer.

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September 22, 2019

A chromosome conformation capture ordered sequence of the barley genome.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

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September 22, 2019

The transcriptome of human pluripotent stem cells.

Human Embryonic Stem Cells (hESCs) are in vitro derivatives of the inner cell mass of the blastocyst and are characterized by an undifferentiated and pluripotent state that can be perpetuated in time, indefinitely. hESCs provide a unique opportunity to both dissect the molecular mechanisms that are predisposed to the maintenance of pluripotency and model the ability to initiate differentiation and cell commitment within the developing embryo. To fully understand these mechanisms, it is necessary to accurately identify the specific transcriptome of hESCs. Many distinct gene annotation methods, such as cDNA and EST sequencing and RNA-Seq, have been used to identify the transcriptome of hESCs. Lately, we developed a new tool (IDP) to integrate the hybrid sequencing data to characterize a more reliable and comprehensive hESC transcriptome with discoveries of many novel transcripts. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

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September 22, 2019

Cow, yak, and camel milk diets differentially modulated the systemic immunity and fecal microbiota of rats

Cow milk is most widely consumed; however, non-cattle milk has gained increasing interest because of added nutritive values. We compared the health effects of yak, cow, and camel milk in rats. By measuring several plasma immune factors, significantly more interferon-? was detected in the camel than the yak (P=0.0020) or cow (P=0.0062) milk group. Significantly more IgM was detected in the yak milk than the control group (P=0.0071). The control group had significantly less interleukin 6 than the yak (P=0.0499) and cow (P=0.0248) milk groups. The fecal microbiota of the 144 samples comprised mainly of the Firmicutes (76.70±11.03%), Bacteroidetes (15.27±7.79%), Proteobacteria (3.61±4.34%), and Tenericutes (2.61±2.53%) phyla. Multivariate analyses revealed a mild shift in the fecal microbiota along the milk treatment. We further identified the differential microbes across the four groups. At day 14, 22 and 28 differential genera and species were identified (P=0.0000–0.0462), while 8 and 11 differential genera and species (P=0.0000–0.0013) were found at day 28. Some short-chain fatty acid and succinate producers increased, while certain health-concerned bacteria (Prevotella copri, Phascolarctobacterium faecium, and Bacteroides uniformis) decreased after 14days of yak or camel milk treatment. We demonstrated that different animal milk could confer distinctive nutritive value to the host.

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September 22, 2019

Transcriptome profiling in the spathe of Anthurium andraeanum ‘Albama’ and its anthocyanin-loss mutant ‘Xueyu’.

Anthurium andraeanum is a popular tropical ornamental plant. Its spathes are brilliantly coloured due to variable anthocyanin contents. To examine the mechanisms that control anthocyanin biosynthesis, we sequenced the spathe transcriptomes of ‘Albama’, a red-spathed cultivar of A. andraeanum, and ‘Xueyu’, its anthocyanin-loss mutant. Both long reads and short reads were sequenced. Long read sequencing produced 805,869 raw reads, resulting in 83,073 high-quality transcripts. Short read sequencing produced 347.79?M reads, and the subsequent assembly resulted in 111,674 unigenes. High-quality transcripts and unigenes were quantified using the short reads, and differential expression analysis was performed between ‘Albama’ and ‘Xueyu’. Obtaining high-quality, full-length transcripts enabled the detection of long transcript structures and transcript variants. These data provide a foundation to elucidate the mechanisms regulating the biosynthesis of anthocyanin in A. andraeanum.

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September 22, 2019

Leveraging multiple transcriptome assembly methods for improved gene structure annotation.

The performance of RNA sequencing (RNA-seq) aligners and assemblers varies greatly across different organisms and experiments, and often the optimal approach is not known beforehand.Here, we show that the accuracy of transcript reconstruction can be boosted by combining multiple methods, and we present a novel algorithm to integrate multiple RNA-seq assemblies into a coherent transcript annotation. Our algorithm can remove redundancies and select the best transcript models according to user-specified metrics, while solving common artifacts such as erroneous transcript chimerisms.We have implemented this method in an open-source Python3 and Cython program, Mikado, available on GitHub.

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September 22, 2019

PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives.

Second-generation, high-throughput sequencing methods have greatly improved our understanding of the ecology of soil microorganisms, yet the short barcodes (< 500 bp) provide limited taxonomic and phylogenetic information for species discrimination and taxonomic assignment. Here, we utilized the third-generation Pacific Biosciences (PacBio) RSII and Sequel instruments to evaluate the suitability of full-length internal transcribed spacer (ITS) barcodes and longer rRNA gene amplicons for metabarcoding Fungi, Oomycetes and other eukaryotes in soil samples. Metabarcoding revealed multiple errors and biases: Taq polymerase substitution errors and mis-incorporating indels in sequencing homopolymers constitute major errors; sequence length biases occur during PCR, library preparation, loading to the sequencing instrument and quality filtering; primer-template mismatches bias the taxonomic profile when using regular and highly degenerate primers. The RSII and Sequel platforms enable the sequencing of amplicons up to 3000 bp, but the sequence quality remains slightly inferior to Illumina sequencing especially in longer amplicons. The full ITS barcode and flanking rRNA small subunit gene greatly improve taxonomic identification at the species and phylum levels, respectively. We conclude that PacBio sequencing provides a viable alternative for metabarcoding of organisms that are of relatively low diversity, require > 500-bp barcode for reliable identification or when phylogenetic approaches are intended.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019

An environmental bacterial taxon with a large and distinct metabolic repertoire.

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such ‘talented’ producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus ‘Entotheonella’ with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. ‘Entotheonella’ spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum ‘Tectomicrobia’. The pronounced bioactivities and chemical uniqueness of ‘Entotheonella’ compounds provide significant opportunities for ecological studies and drug discovery.

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September 22, 2019

A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation.

Alternative splicing (AS) is a crucial regulatory mechanism in eukaryotes, which acts by greatly increasing transcriptome diversity. The extent and complexity of AS has been revealed in model plants using high-throughput next-generation sequencing. However, this technique is less effective in accurately identifying transcript isoforms in polyploid species because of the high sequence similarity between coexisting subgenomes. Here we characterize AS in the polyploid species cotton. Using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq), we developed an integrated pipeline for Iso-Seq transcriptome data analysis (https://github.com/Nextomics/pipeline-for-isoseq). We identified 176 849 full-length transcript isoforms from 44 968 gene models and updated gene annotation. These data led us to identify 15 102 fibre-specific AS events and estimate that c. 51.4% of homoeologous genes produce divergent isoforms in each subgenome. We reveal that AS allows differential regulation of the same gene by miRNAs at the isoform level. We also show that nucleosome occupancy and DNA methylation play a role in defining exons at the chromatin level. This study provides new insights into the complexity and regulation of AS, and will enhance our understanding of AS in polyploid species. Our methodology for Iso-Seq data analysis will be a useful reference for the study of AS in other species.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019

Transcriptional adaptations during long-term persistence of Staphylococcus aureus in the airways of a cystic fibrosis patient.

The lungs of Cystic fibrosis (CF) patients are often colonized and/or infected by Staphylococcus aureus for years, mostly by one predominant clone. For long-term survival in this environment, S. aureus needs to adapt during its interactions with host factors, antibiotics, and other pathogens. Here, we study long-term transcriptional as well as genomic adaptations of an isogenic pair of S. aureus isolates from a single patient using RNA sequencing (RNA-Seq) and whole genome sequencing (WGS). Mimicking in vivo conditions, we cultivated the S. aureus isolates using artificial sputum medium before harvesting RNA for subsequent analysis. We confirmed our RNA-Seq data using quantitative real-time (qRT)-PCR and additionally investigated intermediate isolates from the same patient representing in total 13.2 years of persistence in the CF airways. Comparative RNA-Seq analysis of the first and the last (“late”) isolate revealed significant differences in the late isolate after 13.2 years of persistence. Of the 2545 genes expressed in both isolates that were cultivated aerobically, 256 genes were up- and 161 were down-regulated with a minimum 2-fold change (2f). Focusing on 25 highly (=8f) up- (n=9) or down- (n=16) regulated genes, we identified several genes encoding for virulence factors involved in immune evasion, bacterial spread or secretion (e.g. spa, sak, and esxA). Moreover, these genes displayed similar expression trends under aerobic, microaerophilic and anaerobic conditions. Further qRT-PCR-experiments of highly up- or down-regulated genes within intermediate S. aureus isolates resulted in different gene expression patterns over the years. Using sequencing analysis of the differently expressed genes and their upstream regions in the late S. aureus isolate resulted in only few genomic alterations. Comparative transcriptomic analysis revealed adaptive changes affecting mainly genes involved in host-pathogen interaction. Although the underlying mechanisms were not known, our results suggest adaptive processes beyond genomic mutations triggered by local factors rather than by activation of global regulators. Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.

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September 22, 2019

Assessing quality of Medicago sativa silage by monitoring bacterial composition with single molecule, real-time sequencing technology and various physiological parameters.

The present study applied the PacBio single molecule, real-time sequencing technology (SMRT) in evaluating the quality of silage production. Specifically, we produced four types of Medicago sativa silages by using four different lactic acid bacteria-based additives (AD-I, AD-II, AD-III and AD-IV). We monitored the changes in pH, organic acids (including butyric acid, the ratio of acetic acid/lactic acid, ?-aminobutyric acid, 4-hyroxy benzoic acid and phenyl lactic acid), mycotoxins, and bacterial microbiota during silage fermentation. Our results showed that the use of the additives was beneficial to the silage fermentation by enhancing a general pH and mycotoxin reduction, while increasing the organic acids content. By SMRT analysis of the microbial composition in eight silage samples, we found that the bacterial species number and relative abundances shifted apparently after fermentation. Such changes were specific to the LAB species in the additives. Particularly, Bacillus megaterium was the initial dominant species in the raw materials; and after the fermentation process, Pediococcus acidilactici and Lactobacillus plantarum became the most prevalent species, both of which were intrinsically present in the LAB additives. Our data have demonstrated that the SMRT sequencing platform is applicable in assessing the quality of silage.

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September 22, 2019

Comparison of the mitochondrial genomes and steady state transcriptomes of two strains of the trypanosomatid parasite, Leishmania tarentolae.

U-insertion/deletion RNA editing is a post-transcriptional mitochondrial RNA modification phenomenon required for viability of trypanosomatid parasites. Small guide RNAs encoded mainly by the thousands of catenated minicircles contain the information for this editing. We analyzed by NGS technology the mitochondrial genomes and transcriptomes of two strains, the old lab UC strain and the recently isolated LEM125 strain. PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions. Minicircles were identified by a characteristic size, the presence of three short conserved sequences, a region of inherently bent DNA and the presence of single gRNA genes at a fairly defined location. The LEM125 strain contained over 114 minicircles encoding different gRNAs and the UC strain only ~24 minicircles. Some LEM125 minicircles contained no identifiable gRNAs. Approximate copy numbers of the different minicircle classes in the network were determined by the number of PacBio CCS reads that assembled to each class. Mitochondrial RNA libraries from both strains were mapped against the minicircle and maxicircle sequences. Small RNA reads mapped to the putative gRNA genes but also to multiple regions outside the genes on both strands and large RNA reads mapped in many cases over almost the entire minicircle on both strands. These data suggest that minicircle transcription is complete and bidirectional, with 3′ processing yielding the mature gRNAs. Steady state RNAs in varying abundances are derived from all maxicircle genes, including portions of the repetitive divergent region. The relative extents of editing in both strains correlated with the presence of a cascade of cognate gRNAs. These data should provide the foundation for a deeper understanding of this dynamic genetic system as well as the evolutionary variation of editing in different strains.

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September 22, 2019

Improving PacBio long read accuracy by short read alignment.

The recent development of third generation sequencing (TGS) generates much longer reads than second generation sequencing (SGS) and thus provides a chance to solve problems that are difficult to study through SGS alone. However, higher raw read error rates are an intrinsic drawback in most TGS technologies. Here we present a computational method, LSC, to perform error correction of TGS long reads (LR) by SGS short reads (SR). Aiming to reduce the error rate in homopolymer runs in the main TGS platform, the PacBio® RS, LSC applies a homopolymer compression (HC) transformation strategy to increase the sensitivity of SR-LR alignment without scarifying alignment accuracy. We applied LSC to 100,000 PacBio long reads from human brain cerebellum RNA-seq data and 64 million single-end 75 bp reads from human brain RNA-seq data. The results show LSC can correct PacBio long reads to reduce the error rate by more than 3 folds. The improved accuracy greatly benefits many downstream analyses, such as directional gene isoform detection in RNA-seq study. Compared with another hybrid correction tool, LSC can achieve over double the sensitivity and similar specificity.

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