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September 22, 2019

Tracking alternatively spliced isoforms from long reads by SpliceHunter.

Alternative splicing increases the functional complexity of a genome by generating multiple isoforms and potentially proteins from the same gene. Vast amounts of alternative splicing events are routinely detected by short read deep sequencing technologies but their functional interpretation is hampered by an uncertain transcript context. Emerging long-read sequencing technologies provide a more complete picture of full-length transcript sequences. We introduce SpliceHunter, a tool for the computational interpretation of long reads generated by for example Pacific Biosciences instruments. SpliceHunter defines and tracks isoforms and novel transcription units across time points, compares their splicing pattern to a reference annotation, and translates them into potential protein sequences.

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September 22, 2019

PacBio sequencing and its applications.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

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September 22, 2019

Sequence of the sugar pine megagenome.

Until very recently, complete characterization of the megagenomes of conifers has remained elusive. The diploid genome of sugar pine (Pinus lambertiana Dougl.) has a highly repetitive, 31 billion bp genome. It is the largest genome sequenced and assembled to date, and the first from the subgenus Strobus, or white pines, a group that is notable for having the largest genomes among the pines. The genome represents a unique opportunity to investigate genome “obesity” in conifers and white pines. Comparative analysis of P. lambertiana and P. taeda L. reveals new insights on the conservation, age, and diversity of the highly abundant transposable elements, the primary factor determining genome size. Like most North American white pines, the principal pathogen of P. lambertiana is white pine blister rust (Cronartium ribicola J.C. Fischer ex Raben.). Identification of candidate genes for resistance to this pathogen is of great ecological importance. The genome sequence afforded us the opportunity to make substantial progress on locating the major dominant gene for simple resistance hypersensitive response, Cr1 We describe new markers and gene annotation that are both tightly linked to Cr1 in a mapping population, and associated with Cr1 in unrelated sugar pine individuals sampled throughout the species’ range, creating a solid foundation for future mapping. This genomic variation and annotated candidate genes characterized in our study of the Cr1 region are resources for future marker-assisted breeding efforts as well as for investigations of fundamental mechanisms of invasive disease and evolutionary response. Copyright © 2016 by the Genetics Society of America.

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September 22, 2019

Transcriptome characterization of moso bamboo (Phyllostachys edulis) seedlings in response to exogenous gibberellin applications.

Moso bamboo (Phyllostachys edulis) is a well-known bamboo species of high economic value in the textile industry due to its rapid growth. Phytohormones, which are master regulators of growth and development, serve as important endogenous signals. However, the mechanisms through which phytohormones regulate growth in moso bamboo remain unknown to date.Here, we reported that exogenous gibberellins (GA) applications resulted in a significantly increased internode length and lignin condensation. Transcriptome sequencing revealed that photosynthesis-related genes were enriched in the GA-repressed gene class, which was consistent with the decrease in leaf chlorophyll concentrations and the lower rate of photosynthesis following GA treatment. Exogenous GA applications on seedlings are relatively easy to perform, thus we used 4-week-old whole seedlings of bamboo for GA- treatment followed by high throughput sequencing. In this study, we identified 932 cis-nature antisense transcripts (cis-NATs), and 22,196 alternative splicing (AS) events in total. Among them, 42 cis-nature antisense transcripts (cis-NATs) and 442 AS events were differentially expressed upon exposure to exogenous GA3, suggesting that post-transcriptional regulation might be also involved in the GA3 response. Targets of differential expression of cis-NATs included genes involved in hormone receptor, photosynthesis and cell wall biogenesis. For example, LAC4 and its corresponding cis-NATs were GA3-induced, and may be involved in the accumulation of lignin, thus affecting cell wall composition.This study provides novel insights illustrating how GA alters post-transcriptional regulation and will shed light on the underlying mechanism of growth modulated by GA in moso bamboo.

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September 22, 2019

Revealing the transcriptomic complexity of switchgrass by PacBio long-read sequencing.

Switchgrass (Panicum virgatum L.) is an important bioenergy crop widely used for lignocellulosic research. While extensive transcriptomic analyses have been conducted on this species using short read-based sequencing techniques, very little has been reliably derived regarding alternatively spliced (AS) transcripts.We present an analysis of transcriptomes of six switchgrass tissue types pooled together, sequenced using Pacific Biosciences (PacBio) single-molecular long-read technology. Our analysis identified 105,419 unique transcripts covering 43,570 known genes and 8795 previously unknown genes. 45,168 are novel transcripts of known genes. A total of 60,096 AS transcripts are identified, 45,628 being novel. We have also predicted 1549 transcripts of genes involved in cell wall construction and remodeling, 639 being novel transcripts of known cell wall genes. Most of the predicted transcripts are validated against Illumina-based short reads. Specifically, 96% of the splice junction sites in all the unique transcripts are validated by at least five Illumina reads. Comparisons between genes derived from our identified transcripts and the current genome annotation revealed that among the gene set predicted by both analyses, 16,640 have different exon-intron structures.Overall, substantial amount of new information is derived from the PacBio RNA data regarding both the transcriptome and the genome of switchgrass.

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September 22, 2019

A carnivorous plant genetic map: pitcher/insect-capture QTL on a genetic linkage map of Sarracenia.

The study of carnivorous plants can afford insight into their unique evolutionary adaptations and their interactions with prokaryotic and eukaryotic species. For Sarracenia (pitcher plants), we identified 64 quantitative trait loci (QTL) for insect-capture traits of the pitchers, providing the genetic basis for differences between the pitfall and lobster-trap strategies of insect capture. The linkage map developed here is based upon the F2 of a cross between Sarracenia rosea and Sarracenia psittacina; we mapped 437 single nucleotide polymorphism and simple sequence repeat markers. We measured pitcher traits which differ between S. rosea and S. psittacina, mapping 64 QTL for 17 pitcher traits; there are hot-spot locations where multiple QTL map near each other. There are epistatic interactions in many cases where there are multiple loci for a trait. The QTL map uncovered the genetic basis for the differences between pitfall- and lobster-traps, and the changes that occurred during the divergence of these species. The longevity and clonability of Sarracenia plants make the F2 mapping population a resource for mapping more traits and for phenotype-to-genotype studies.

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September 22, 2019

Analysis of transcripts and splice isoforms in red clover (Trifolium pratense L.) by single-molecule long-read sequencing.

Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and completed structure of mRNA transcripts remain unclear, which limit further explore on red clover.In this study, the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length splice isoforms, and 29,730 novel isoforms from known genes and 2194 novel isoforms from novel genes were identified. A total of 5492 alternative splicing events was identified and the majority of alter spliced events in red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8719 including 186,517 transcripts have at least one poly(A) site. Furthermore, we identified 4333 long non-coding RNAs and 3762 fusion transcripts.We analyzed full-length transcriptome of red clover with PacBio SMRT. Those new findings provided important information for improving red clover draft genome annotation and fully characterization of red clover transcriptome.

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September 22, 2019

Long-read sequencing and de novo assembly of a Chinese genome.

Short-read sequencing has enabled the de novo assembly of several individual human genomes, but with inherent limitations in characterizing repeat elements. Here we sequence a Chinese individual HX1 by single-molecule real-time (SMRT) long-read sequencing, construct a physical map by NanoChannel arrays and generate a de novo assembly of 2.93?Gb (contig N50: 8.3?Mb, scaffold N50: 22.0?Mb, including 39.3?Mb N-bases), together with 206?Mb of alternative haplotypes. The assembly fully or partially fills 274 (28.4%) N-gaps in the reference genome GRCh38. Comparison to GRCh38 reveals 12.8?Mb of HX1-specific sequences, including 4.1?Mb that are not present in previously reported Asian genomes. Furthermore, long-read sequencing of the transcriptome reveals novel spliced genes that are not annotated in GENCODE and are missed by short-read RNA-Seq. Our results imply that improved characterization of genome functional variation may require the use of a range of genomic technologies on diverse human populations.

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September 22, 2019

Deciphering highly similar multigene family transcripts from Iso-Seq data with IsoCon

A significant portion of genes in vertebrate genomes belongs to multigene families, with each family containing several gene copies whose presence/absence, as well as isoform structure, can be highly variable across individuals. Existing de novo techniques for assaying the sequences of such highly-similar gene families fall short of reconstructing end-to-end transcripts with nucleotide-level precision or assigning alternatively spliced transcripts to their respective gene copies. We present IsoCon, a high-precision method using long PacBio Iso-Seq reads to tackle this challenge. We apply IsoCon to nine Y chromosome ampliconic gene families and show that it outperforms existing methods on both experimental and simulated data. IsoCon has allowed us to detect an unprecedented number of novel isoforms and has opened the door for unraveling the structure of many multigene families and gaining a deeper understanding of genome evolution and human diseases.

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September 22, 2019

Construction of a draft reference transcripts of onion (Allium cepa) using long-read sequencing

To obtain intact and full-length RNA transcripts of onion (Allium cepa), long-read sequencing technology was first applied. Total RNAs extracted from four tissues; flowers, leaves, bulbs and roots, of red–purple and yellow-colored onions (A. cepa) were sequenced using long-read sequencing (RSII platform, P4-C2 chemistry). The 99,247 polished high-quality isoforms were produced by sequence correction processes of consensus calling, quality filtering, orientation verification, misread-nucleotide correction and dot-matrix view. The dot-matrix view was subsequently used to remove artificial inverted repeats (IRs), and resultantly 421 IRs were removed. The remaining 98,826 isoforms were condensed to 35,505 through the removal process of redundant isoforms. To assess the completeness of the 35,505 isoforms, the ratio of full-length isoforms, short-read mapping to the isoforms, and differentially expressed genes among the four tissues were analyzed along with the gene ontology across the tissues. As a result, the 35,505 isoforms were verified as a collection of isoforms with high completeness, and designated as draft reference transcripts (DRTs, ver 1.0) constructed by long-read sequencing.

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September 22, 2019

Transcriptome-wide survey of pseudorabies virus using next- and third-generation sequencing platforms.

Pseudorabies virus (PRV) is an alphaherpesvirus of swine. PRV has a large double-stranded DNA genome and, as the latest investigations have revealed, a very complex transcriptome. Here, we present a large RNA-Seq dataset, derived from both short- and long-read sequencing. The dataset contains 1.3 million 100?bp paired-end reads that were obtained from the Illumina random-primed libraries, as well as 10 million 50?bp single-end reads generated by the Illumina polyA-seq. The Pacific Biosciences RSII non-amplified method yielded 57,021 reads of inserts (ROIs) aligned to the viral genome, the amplified method resulted in 158,396 PRV-specific ROIs, while we obtained 12,555 ROIs using the Sequel platform. The Oxford Nanopore’s MinION device generated 44,006 reads using their regular cDNA-sequencing method, whereas 29,832 and 120,394 reads were produced by using the direct RNA-sequencing and the Cap-selection protocols, respectively. The raw reads were aligned to the PRV reference genome (KJ717942.1). Our provided dataset can be used to compare different sequencing approaches, library preparation methods, as well as for validation and testing bioinformatic pipelines.

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September 22, 2019

De novo assembly and characterizing of the culm-derived meta-transcriptome from the polyploid sugarcane genome based on coding transcripts

Sugarcane biomass has been used for sugar, bioenergy and biomaterial production. The majority of the sugarcane biomass comes from the culm, which makes it important to understand the genetic control of biomass production in this part of the plant. A meta-transcriptome of the culm was obtained in an earlier study by using about one billion paired-end (150 bp) reads of deep RNA sequencing of samples from 20 diverse sugarcane genotypes and combining de novo assemblies from different assemblers and different settings. Although many genes could be recovered, this resulted in a large combined assembly which created the need for clustering to reduce transcript redundancy while maintaining gene content. Here, we present a comprehensive analysis of the effect of different assembly settings and clustering methods on de novo assembly, annotation and transcript profiling focusing especially on the coding transcripts from the highly polyploid sugarcane genome. The new coding sequence-based transcript clustering resulted in a better representation of transcripts compared to the earlier approach, having 121,987 contigs, which included 78,052 main and 43,935 alternative transcripts. About 73%, 67%, 61% and 10% of the transcriptome was annotated against the NCBI NR protein database, GO terms, orthologous groups and KEGG orthologies, respectively. Using this set for a differential gene expression analysis between the young and mature sugarcane culm tissues, a total of 822 transcripts were found to be differentially expressed, including key transcripts involved in sugar/fiber accumulation in sugarcane. In the context of the lack of a whole genome sequence for sugarcane, the availability of a well annotated culm-derived meta-transcriptome through deep sequencing provides useful information on coding genes specific to the sugarcane culm and will certainly contribute to understanding the process of carbon partitioning, and biomass accumulation in the sugarcane culm.

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September 22, 2019

Long-read sequencing of the human cytomegalovirus transcriptome with the Pacific Biosciences RSII platform.

Long-read RNA sequencing allows for the precise characterization of full-length transcripts, which makes it an indispensable tool in transcriptomics. The human cytomegalovirus (HCMV) genome has been first sequenced in 1989 and although short-read sequencing studies have uncovered much of the complexity of its transcriptome, only few of its transcripts have been fully annotated. We hereby present a long-read RNA sequencing dataset of HCMV infected human lung fibroblast cells sequenced by the Pacific Biosciences RSII platform. Seven SMRT cells were sequenced using oligo(dT) primers to reverse transcribe poly(A)-selected RNA molecules and one library was prepared using random primers for the reverse transcription of the rRNA-depleted sample. Our dataset contains 122,636 human and 33,086 viral (HMCV strain Towne) reads. The described data include raw and processed sequencing files, and combined with other datasets, they can be used to validate transcriptome analysis tools, to compare library preparation methods, to test base calling algorithms or to identify genetic variants.

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September 22, 2019

Comparative transcriptomic and physiological analyses of Medicago sativa L. indicates that multiple regulatory networks are activated during continuous ABA treatment.

Alfalfa is the most extensively cultivated forage legume worldwide. However, the molecular mechanisms underlying alfalfa responses to exogenous abscisic acid (ABA) are still unknown. In this study, the first global transcriptome profiles of alfalfa roots under ABA treatments for 1, 3 and 12 h (three biological replicates for each time point, including the control group) were constructed using a BGISEQ-500 sequencing platform. A total of 50,742 isoforms with a mean length of 2541 bp were generated, and 4944 differentially expressed isoforms (DEIs) were identified after ABA deposition. Metabolic analyses revealed that these DEIs were involved in plant hormone signal transduction, transcriptional regulation, antioxidative defense and pathogen immunity. Notably, several well characterized hormone signaling pathways, for example, the core ABA signaling pathway, was activated, while salicylic acid, jasmonate and ethylene signaling pathways were mainly suppressed by exogenous ABA. Moreover, the physiological work showed that catalase and peroxidase activity and glutathione and proline content were increased after ABA deposition, which is in accordance with the dynamic transcript profiles of the relevant genes in antioxidative defense system. These results indicate that ABA has the potential to improve abiotic stress tolerance, but that it may negatively regulate pathogen resistance in alfalfa.

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September 22, 2019

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome.

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.

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