RNA-seq Assembly Research Articles

CloudrnaSPAdes: isoform assembly using bulk barcoded RNA sequencing data.

Recent advancements in long-read RNA sequencing have enabled the examination of full-length isoforms, previously uncaptured by short-read sequencing methods. An alternative powerful method for studying isoforms is through the use of barcoded short-read RNA reads, for which a barcode indicates whether two short-reads arise from the same molecule or not. Such techniques included the 10x Genomics linked-read based SParse Isoform Sequencing (SPIso-seq), as well as Loop-Seq, or Tell-Seq. Some applications, such as novel-isoform discovery, require very high coverage. Obtaining high coverage using long reads can be difficult, making barcoded RNA-seq data a valuable alternative for this task. However, most annotation pipelines are not able to work with a set of short reads instead of a single transcript, also not able to work with coverage gaps within a molecule if any. In order to overcome this challenge, we present an RNA-seq assembler that allows the determination of the expressed isoform per barcode. In this article, we present cloudrnaSPAdes, a tool for assembling full-length isoforms from barcoded RNA-seq linked-read data in a reference-free fashion. Evaluating it on simulated and real human data, we found that cloudrnaSPAdes accurately assembles isoforms, even for genes with high isoform diversity. cloudrnaSPAdes is a feature release of a SPAdes assembler and version used for this article is available at https://github.com/1dayac/cloudrnaSPAdes-release.

Deciphering the enigma of RNA editing in the ATP1_alpha subunit of ATP synthase in Triticum aestivum

There is evidence that RNA editing is related to plant cellular stress as well as electron transport organelles, such as mitochondria. The mitochondrial atp1 gene encodes the alpha-subunit of Atp synthase. Control as well as two periods of drought stress treatments were analyzed in the cDNAs generated from the mitochondrial atp1 gene of two cultivars of Triticum aestivum [Giza 168 (G168) and Gemmiza 10 (GM10)]. Following RNA-seq data assembly, atp1 cDNAs from the control (acc. no. OQ129415), 2-hour (acc. no. OQ129416), and 12-hour (acc. no. OQ129417) time points of the T. aestivum cultivar G168 were obtained. Control (acc. no. OQ129419), 2-hour (acc. no. OQ129420), and 12-hour (acc. no. OQ129421) samples all included reconstructed atp1 transcripts from Gemmiza 10. Atp1 transcripts were assembled using the wheat atp1 gene (acc. no. NC_036024). RNA-seq raw data was utilized to identify 11 RNA editing sites in atp1 in the tolerant cultivar Giza168 and 6 in the sensitive cultivar Gemmiza10. The significant difference in RNA editing observed between control and drought stress conditions in sites led to synonymous amino acids. This led to no change in tertiary structure between tolerant and sensitive cultivars. But the change was focused between produced protein and its correspondence sequence on DNA.

Comparative expression analysis of water buffalo (Bubalus bubalis) to identify genes associated with economically important traits.

The milk, meat, skins, and draft power of domestic water buffalo (Bubalus bubalis) provide substantial contributions to the global agricultural economy. The world's water buffalo population is primarily found in Asia, and the buffalo supports more people per capita than any other livestock species. For evaluating the workflow, output rate, and completeness of transcriptome assemblies within and between reference-free (RF) de novo transcriptome and reference-based (RB) datasets, abundant bioinformatics studies have been carried out to date. However, comprehensive documentation of the degree of consistency and variability of the data produced by comparing gene expression levels using these two separate techniques is lacking. In the present study, we assessed the variations in the number of differentially expressed genes (DEGs) attained with RF and RB approaches. In light of this, we conducted a study to identify, annotate, and analyze the genes associated with four economically important traits of buffalo, viz., milk volume, age at first calving, post-partum cyclicity, and feed conversion efficiency. A total of 14,201 and 279 DEGs were identified in RF and RB assemblies. Gene ontology (GO) terms associated with the identified genes were allocated to traits under study. Identified genes improve the knowledge of the underlying mechanism of trait expression in water buffalo which may support improved breeding plans for higher productivity. The empirical findings of this study using RNA-seq data-based assembly may improve the understanding of genetic diversity in relation to buffalo productivity and provide important contributions to answer biological issues regarding the transcriptome of non-model organisms.

First characterization of PIWI-interacting RNA clusters in a cichlid fish with a B chromosome

BackgroundB chromosomes are extra elements found in several eukaryote species. Usually, they do not express a phenotype in the host. However, advances in bioinformatics over the last decades have allowed us to describe several genes and molecular functions related to B chromosomes. These advances enable investigations of the relationship between the B chromosome and the host to understand how this element has been preserved in genomes. However, considering that transposable elements (TEs) are highly abundant in this supernumerary chromosome, there is a lack of knowledge concerning the dynamics of TE control in B-carrying cells. Thus, the present study characterized PIWI-interacting RNA (piRNA) clusters and pathways responsible for silencing the mobilization of TEs in gonads of the cichlid fish Astatotilapia latifasciata carrying the B chromosome.ResultsThrough small RNA-seq and genome assembly, we predicted and annotated piRNA clusters in the A. latifasciata genome for the first time. We observed that these clusters had biased expression related to sex and the presence of the B chromosome. Furthermore, three piRNA clusters, named curupira, were identified in the B chromosome. Two of them were expressed exclusively in gonads of samples with the B chromosome. The composition of these curupira sequences was derived from LTR, LINE, and DNA elements, representing old and recent transposition events in the A. latifasciata genome and the B chromosome. The presence of the B chromosome also affected the expression of piRNA pathway genes. The mitochondrial cardiolipin hydrolase-like (pld6) gene is present in the B chromosome, as previously reported, and an increase in its expression was detected in gonads with the B chromosome.ConclusionsDue to the high abundance of TEs in the B chromosome, it was possible to investigate the origin of piRNA from these jumping genes. We hypothesize that the B chromosome has evolved its own genomic guardians to prevent uncontrolled TE mobilization. Furthermore, we also detected an expression bias in the presence of the B chromosome over A. latifasciata piRNA clusters and pathway genes.

Antennal transcriptome analysis of chemosensory genes in the cowpea beetle, Callosobruchus maculatus (F.).

Olfaction, one of the most important sensory systems governing insect behavior, is a possible target for pest management. Therefore, in this study, we analyzed the antennal transcriptome of the cowpea beetle, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae: Bruchinae), which is a major pest of stored pulses and legumes. The de novo antennal RNA-seq assembly results identified 17 odorant, 2 gustatory, and 10 ionotropic receptors, 1 sensory neuron membrane protein, and 12 odorant-binding and 7 chemosensory proteins. Moreover, differential gene expression analysis of virgin male and female antennal samples followed by qRT-PCR revealed 1 upregulated and 4 downregulated odorant receptors in males. We also performed homology searches using the coding sequences built from previously proposed amino acid sequences derived from genomic data and identified additional chemosensory-related genes.

Root transcriptome of Allium mongolicum Regel under drought stress conditions

Allium mongolicum Regel is a desert plant that shows great resistance to wind erosion, drought, and low temperatures and is widely distributed in the desert lands of China. Understanding the molecular mechanisms underlying its drought resistance is essential for uncovering drought resistance genes and improving its beneficial traits. Here, a de novo RNA-Seq assembly analysis was conducted using the roots of 1-month-old seedlings under drought stress. Using pairwise comparisons of untreated plants (CK), 48-hour drought plants (G48h), and 96- hour drought plants (G96h), in total, 2,211 differentially expressed unigenes (DEGs) were obtained. Furthermore, using functional annotation, these DEGs were mainly involved in the plasma membrane, photosynthesis, and secondary carbohydrate metabolism. Moreover, genes involved in the ABA-mediated signaling pathway and secondary metabolism were upregulated in the roots. These results suggest that desert plants may use signaling and secondary metabolic pathways as adaptive responses to drought stress. Collectively, this work could help elucidate the molecular mechanisms underlying the ability of A. mongolicum Regel to respond to drought stress and aid in the selection of novel drought tolerance genes.

RNASeq analysis of drought-stressed guayule reveals the role of gene transcription for modulating rubber, resin, and carbohydrate synthesis

The drought-adapted shrub guayule (Parthenium argentatum) produces rubber, a natural product of major commercial importance, and two co-products with potential industrial use: terpene resin and the carbohydrate fructan. The rubber content of guayule plants subjected to water stress is higher compared to that of well-irrigated plants, a fact consistently reported in guayule field evaluations. To better understand how drought influences rubber biosynthesis at the molecular level, a comprehensive transcriptome database was built from drought-stressed guayule stem tissues using de novo RNA-seq and genome-guided assembly, followed by annotation and expression analysis. Despite having higher rubber content, most rubber biosynthesis related genes were down-regulated in drought-stressed guayule, compared to well-irrigated plants, suggesting post-transcriptional effects may regulate drought-induced rubber accumulation. On the other hand, terpene resin biosynthesis genes were unevenly affected by water stress, implying unique environmental influences over transcriptional control of different terpene compounds or classes. Finally, drought induced expression of fructan catabolism genes in guayule and significantly suppressed these fructan biosynthesis genes. It appears then, that in guayule cultivation, irrigation levels might be calibrated in such a regime to enable tunable accumulation of rubber, resin and fructan.

Ryūtō: improved multi-sample transcript assembly for differential transcript expression analysis and more.

Accurate assembly of RNA-seq is a crucial step in many analytic tasks such as gene annotation or expression studies. Despite ongoing research, progress on traditional single sample assembly has brought no major breakthrough. Multi-sample RNA-Seq experiments provide more information than single sample datasets and thus constitute a promising area of research. Yet, this advantage is challenging to utilize due to the large amount of accumulating errors. We present an extension to Ryūtō enabling the reconstruction of consensus transcriptomes from multiple RNA-seq datasets, incorporating consensus calling at low level features. We report stable improvements already at three replicates. Ryūtō outperforms competing approaches, providing a better and user-adjustable sensitivity-precision trade-off. Ryūtō's unique ability to utilize a (incomplete) reference for multi sample assemblies greatly increases precision. We demonstrate benefits for differential expression analysis.Ryūtō consistently improves assembly on replicates of the same tissue independent of filter settings, even when mixing conditions or time series. Consensus voting in Ryūtō is especially effective at high precision assembly, while Ryūtō's conventional mode can reach higher recall. Ryūtō is available at https://github.com/studla/RYUTO. Supplementary data are available at Bioinformatics online.

De novo transcriptome analysis and comparative expression profiling of genes associated with the taste-modifying protein neoculin in Curculigo latifolia and Curculigo capitulata fruits

BackgroundCurculigo latifolia is a perennial plant endogenous to Southeast Asia whose fruits contain the taste-modifying protein neoculin, which binds to sweet receptors and makes sour fruits taste sweet. Although similar to snowdrop (Galanthus nivalis) agglutinin (GNA), which contains mannose-binding sites in its sequence and 3D structure, neoculin lacks such sites and has no lectin activity. Whether the fruits of C. latifolia and other Curculigo plants contain neoculin and/or GNA family members was unclear.ResultsThrough de novo RNA-seq assembly of the fruits of C. latifolia and the related C. capitulata and detailed analysis of the expression patterns of neoculin and neoculin-like genes in both species, we assembled 85,697 transcripts from C. latifolia and 76,775 from C. capitulata using Trinity and annotated them using public databases. We identified 70,371 unigenes in C. latifolia and 63,704 in C. capitulata. In total, 38.6% of unigenes from C. latifolia and 42.6% from C. capitulata shared high similarity between the two species. We identified ten neoculin-related transcripts in C. latifolia and 15 in C. capitulata, encoding both the basic and acidic subunits of neoculin in both plants. We aligned these 25 transcripts and generated a phylogenetic tree. Many orthologs in the two species shared high similarity, despite the low number of common genes, suggesting that these genes likely existed before the two species diverged. The relative expression levels of these genes differed considerably between the two species: the transcripts per million (TPM) values of neoculin genes were 60 times higher in C. latifolia than in C. capitulata, whereas those of GNA family members were 15,000 times lower in C. latifolia than in C. capitulata.ConclusionsThe genetic diversity of neoculin-related genes strongly suggests that neoculin genes underwent duplication during evolution. The marked differences in their expression profiles between C. latifolia and C. capitulata may be due to mutations in regions involved in transcriptional regulation. Comprehensive analysis of the genes expressed in the fruits of these two Curculigo species helped elucidate the origin of neoculin at the molecular level.

Wheat leaf rust resistance gene Lr13 is a specific Ne2 allele for hybrid necrosis

Wheat leaf rust resistance gene Lr13 is a specific Ne2 allele for hybrid necrosis

Ion channel profiling of the Lymnaea stagnalis ganglia via transcriptome analysis

BackgroundThe pond snail Lymnaea stagnalis (L. stagnalis) has been widely used as a model organism in neurobiology, ecotoxicology, and parasitology due to the relative simplicity of its central nervous system (CNS). However, its usefulness is restricted by a limited availability of transcriptome data. While sequence information for the L. stagnalis CNS transcripts has been obtained from EST libraries and a de novo RNA-seq assembly, the quality of these assemblies is limited by a combination of low coverage of EST libraries, the fragmented nature of de novo assemblies, and lack of reference genome.ResultsIn this study, taking advantage of the recent availability of a preliminary L. stagnalis genome, we generated an RNA-seq library from the adult L. stagnalis CNS, using a combination of genome-guided and de novo assembly programs to identify 17,832 protein-coding L. stagnalis transcripts. We combined our library with existing resources to produce a transcript set with greater sequence length, completeness, and diversity than previously available ones. Using our assembly and functional domain analysis, we profiled L. stagnalis CNS transcripts encoding ion channels and ionotropic receptors, which are key proteins for CNS function, and compared their sequences to other vertebrate and invertebrate model organisms. Interestingly, L. stagnalis transcripts encoding numerous putative Ca2+ channels showed the most sequence similarity to those of Mus musculus, Danio rerio, Xenopus tropicalis, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that many calcium channel-related signaling pathways may be evolutionarily conserved.ConclusionsOur study provides the most thorough characterization to date of the L. stagnalis transcriptome and provides insights into differences between vertebrates and invertebrates in CNS transcript diversity, according to function and protein class. Furthermore, this study provides a complete characterization of the ion channels of Lymnaea stagnalis, opening new avenues for future research on fundamental neurobiological processes in this model system.

Hybrid transcriptome sequencing approach improved assembly and gene annotation in Cynara cardunculus (L.)

BackgroundThe investigation of transcriptome profiles using short reads in non-model organisms, which lack of well-annotated genomes, is limited by partial gene reconstruction and isoform detection. In contrast, long-reads sequencing techniques revealed their potential to generate complete transcript assemblies even when a reference genome is lacking. Cynara cardunculus var. altilis (DC) (cultivated cardoon) is a perennial hardy crop adapted to dry environments with many industrial and nutraceutical applications due to the richness of secondary metabolites mostly produced in flower heads. The investigation of this species benefited from the recent release of a draft genome, but the transcriptome profile during the capitula formation still remains unexplored. In the present study we show a transcriptome analysis of vegetative and inflorescence organs of cultivated cardoon through a novel hybrid RNA-seq assembly approach utilizing both long and short RNA-seq reads.ResultsThe inclusion of a single Nanopore flow-cell output in a hybrid sequencing approach determined an increase of 15% complete assembled genes and 18% transcript isoforms respect to short reads alone. Among 25,463 assembled unigenes, we identified 578 new genes and updated 13,039 gene models, 11,169 of which were alternatively spliced isoforms. During capitulum development, 3424 genes were differentially expressed and approximately two-thirds were identified as transcription factors including bHLH, MYB, NAC, C2H2 and MADS-box which were highly expressed especially after capitulum opening. We also show the expression dynamics of key genes involved in the production of valuable secondary metabolites of which capitulum is rich such as phenylpropanoids, flavonoids and sesquiterpene lactones. Most of their biosynthetic genes were strongly transcribed in the flower heads with alternative isoforms exhibiting differentially expression levels across the tissues.ConclusionsThis novel hybrid sequencing approach allowed to improve the transcriptome assembly, to update more than half of annotated genes and to identify many novel genes and different alternatively spliced isoforms. This study provides new insights on the flowering cycle in an Asteraceae plant, a valuable resource for plant biology and breeding in Cynara and an effective method for improving gene annotation.

Transcriptome dataset for RNA-seq analysis of axolotl embryonic oropharyngeal endoderm explants.

Animal nutrition and toxin deterrence rely on the ability to taste, which occurs through columnar taste cells clustered within taste buds. Taste buds in mammals are located within specialized tissues, called papillae. However, taste buds in fish and amphibians, such as axolotls (Ambystoma mexicanum), are not housed in papillae, rather they are embedded within the pharyngeal epithelium. This simplified tissue level organization, along with the ability of cultured oropharyngeal explants from early embryos to produce taste buds on the same time-line as embryos, make the axolotl an excellent model to identify molecules specifically involved in taste bud cell differentiation. We performed de novo transcriptomic analysis on RNA sequences from three different stages of oropharyngeal explants: stages 37/38, 39, and 41. RNA-seq data from 17 total samples representing these stages were pooled to generate a de novo assembly of the transcriptome using a Trinity pipeline. From 27.9Gb of raw sequences, we identified 21,244 transcripts. To our knowledge, this is the first published assembly of axolotl oropharyngeal endoderm explants. This data and transcriptome assembly relate to the research article “Transcriptome Analysis of Axolotl Oropharyngeal Explants During Taste Bud Differentiation Stages” (Kohli et al. 2020). This RNA-seq data and transcriptome assembly provide information on genes expressed in the oropharyngeal endoderm and will be valuable in the identification of taste bud development genes.

Extending rnaSPAdes functionality for hybrid transcriptome assembly

BackgroundDe novo RNA-Seq assembly is a powerful method for analysing transcriptomes when the reference genome is not available or poorly annotated. However, due to the short length of Illumina reads it is usually impossible to reconstruct complete sequences of complex genes and alternative isoforms. Recently emerged possibility to generate long RNA reads, such as PacBio and Oxford Nanopores, may dramatically improve the assembly quality, and thus the consecutive analysis. While reference-based tools for analysing long RNA reads were recently developed, there is no established pipeline for de novo assembly of such data.ResultsIn this work we present a novel method that allows to perform high-quality de novo transcriptome assemblies by combining accuracy and reliability of short reads with exon structure information carried out from long error-prone reads. The algorithm is designed by incorporating existing hybridSPAdes approach into rnaSPAdes pipeline and adapting it for transcriptomic data.ConclusionTo evaluate the benefit of using long RNA reads we selected several datasets containing both Illumina and Iso-seq or Oxford Nanopore Technologies (ONT) reads. Using an existing quality assessment software, we show that hybrid assemblies performed with rnaSPAdes contain more full-length genes and alternative isoforms comparing to the case when only short-read data is used.

Proteogenomics-Guided Evaluation of RNA-Seq Assembly and Protein Database Construction for Emergent Model Organisms.

Proteogenomics is gaining momentum as, today, genomics, transcriptomics, and proteomics can be readily performed on any new species. This approach allows key alterations to molecular pathways to be identified when comparing conditions. For animals and plants, RNA-seq-informed proteomics is the most popular means of interpreting tandem mass spectrometry spectra acquired for species for which the genome has not yet been sequenced. It relies on high-performance de novo RNA-seq assembly and optimized translation strategies. Here, several pre-treatments for Illumina RNA-seq reads before assembly are explored to translate the resulting contigs into useful polypeptide sequences. Experimental transcriptomics and proteomics datasets acquired for individual Gammarus fossarum freshwater crustaceans are used, the most relevant procedure is defined by the ratio of MS/MS spectra assigned to peptide sequences. Removing reads with a mean quality score of less than 17-which represents a single probable nucleotide error on 150-bp reads-prior to assembly, increases the proteomics outcome. The best translation using Transdecoder is achieved with a minimal open reading frame length of 50 amino acids and systematic selection of ORFs longer than 900 nucleotides. Using these parameters, transcriptome assembly and translation informed by proteomics pave the way to further improvements in proteogenomics.

Comparative Transcriptomics Reveals Clues for Differences in Pathogenicity between Hysterothylacium aduncum, Anisakissimplex sensu stricto and Anisakis pegreffii.

Ascaridoid nematodes are widespread in marine fishes. Despite their major socioeconomic importance, mechanisms associated to the fish-borne zoonotic disease anisakiasis are still obscure. RNA-Seq and de-novo assembly were herein applied to RNA extracted from larvae and dissected pharynx of Hysterothylacium aduncum (HA), a non-pathogenic nematode. Assembled transcripts in HA were annotated and compared to the transcriptomes of the zoonotic species Anisakis simplex sensu stricto (AS) and Anisakis pegreffii (AP). Approximately 60,000,000 single-end reads were generated for HA, AS and AP. Transcripts in HA encoded for 30,254 putative peptides while AS and AP encoded for 20,574 and 20,840 putative peptides, respectively. Differential gene expression analyses yielded 471, 612 and 526 transcripts up regulated in the pharynx of HA, AS and AP. The transcriptomes of larvae and pharynx of HA were enriched in transcripts encoding collagen, peptidases, ribosomal proteins and in heat-shock motifs. Transcripts encoding proteolytic enzymes, anesthetics, inhibitors of primary hemostasis and virulence factors, anticoagulants and immunomodulatory peptides were up-regulated in AS and AP pharynx. This study represents the first transcriptomic characterization of a marine parasitic nematode commonly recovered in fish and probably of negligible concern for public health.

IPAC: a genome-guided assembler of isoforms via phasing and combing paths

Full-length transcript reconstruction is very important and quite challenging for the widely used RNA-seq data analysis. Currently available RNA-seq assemblers generally suffered from serious limitations in practical applications, such as low assembly accuracy and incompatibility with latest alignment tools. We introduce iPAC, a new genome-guided assembler for reconstruction of isoforms, which revolutionizes the usage of paired-end and sequencing depth information via phasing and combing paths over a newly designed phasing graph. Tested on both simulated and real datasets, it is to some extent superior to all the salient assemblers of the same kind. Especially, iPAC is significantly powerful in recovery of lowly expressed transcripts while others are not. iPAC is freely available at http://sourceforge.net/projects/transassembly/files. Supplementary data are available at Bioinformatics online.

Nonspecific expression of fertilization genes in the crown-of-thorns Acanthaster cf. solaris: Unexpected evidence of hermaphroditism in a coral reef predator.

The characterization of gene expression in gametes has advanced our understanding of the molecular basis for ecological variation in reproductive success and the evolution of reproductive isolation. These advances are especially significant for ecologically important keystone predators such as the coral-eating crown-of-thorns sea stars (COTS, Acanthaster) which are the most influential predator species in Indo-Pacific coral reef ecosystems and the focus of intensive management efforts. We used RNA-seq and transcriptome assemblies to characterize the expression of genes in mature COTS gonads. We described the sequence and domain organization of eight genes with sex-specific expression and well known functions in fertilization in other echinoderms. We found unexpected expression of genes in one ovary transcriptome that are characteristic of males and sperm, including genes that encode the sperm-specific guanylate cyclase receptor for an egg pheromone, and the sperm acrosomal protein bindin. In a reassembly of previously published RNA-seq data from COTS testes, we found a complementary pattern: strong expression of four genes that are otherwise well known to encode egg-specific fertilization proteins, including the egg receptor for bindin (EBR1) and the acrosome reaction-inducing substance in the egg coat (ARIS1, ARIS2, ARIS3). We also found histological evidence of both eggs and sperm developing in the same gonad in several COTS individuals from a parallel study. These results suggest the occurrence of hermaphrodites, and the potential for reproductive assurance via self-fertilization. Our findings have implications for management of COTS populations, especially in consideration of the large size and massive fecundity of these sea stars.

Cloud accelerated alignment and assembly of full-length single-cell RNA-seq data using Falco

BackgroundRead alignment and transcript assembly are the core of RNA-seq analysis for transcript isoform discovery. Nonetheless, current tools are not designed to be scalable for analysis of full-length bulk or single cell RNA-seq (scRNA-seq) data. The previous version of our cloud-based tool Falco only focuses on RNA-seq read counting, but does not allow for more flexible steps such as alignment and read assembly.ResultsThe Falco framework can harness the parallel and distributed computing environment in modern cloud platforms to accelerate read alignment and transcript assembly of full-length bulk RNA-seq and scRNA-seq data. There are two new modes in Falco: alignment-only and transcript assembly. In the alignment-only mode, Falco can speed up the alignment process by 2.5–16.4x based on two public scRNA-seq datasets when compared to alignment on a highly optimised standalone computer. Furthermore, it also provides a 10x average speed-up compared to alignment using published cloud-enabled tool for read alignment, Rail-RNA. In the transcript assembly mode, Falco can speed up the transcript assembly process by 1.7–16.5x compared to performing transcript assembly on a highly optimised computer.ConclusionFalco is a significantly updated open source big data processing framework that enables scalable and accelerated alignment and assembly of full-length scRNA-seq data on the cloud. The source code can be found at https://github.com/VCCRI/Falco.

Impact of sequencing depth and technology on de novo RNA-Seq assembly

BackgroundRNA-Seq data is inherently nonuniform for different transcripts because of differences in gene expression. This makes it challenging to decide how much data should be generated from each sample. How much should one spend to recover the less expressed transcripts? The sequencing technology used is another consideration, as there are inevitably always biases against certain sequences. To investigate these effects, we first looked at high-depth libraries from a set of well-annotated organisms to ascertain the impact of sequencing depth on de novo assembly. We then looked at libraries sequenced from the Universal Human Reference RNA (UHRR) to compare the performance of Illumina HiSeq and MGI DNBseq™ technologies.ResultsOn the issue of sequencing depth, the amount of exomic sequence assembled plateaued using data sets of approximately 2 to 8 Gbp. However, the amount of genomic sequence assembled did not plateau for many of the analyzed organisms. Most of the unannotated genomic sequences are single-exon transcripts whose biological significance will be questionable for some users. On the issue of sequencing technology, both of the analyzed platforms recovered a similar number of full-length transcripts. The missing “gap” regions in the HiSeq assemblies were often attributed to higher GC contents, but this may be an artefact of library preparation and not of sequencing technology.ConclusionsIncreasing sequencing depth beyond modest data sets of less than 10 Gbp recovers a plethora of single-exon transcripts undocumented in genome annotations. DNBseq™ is a viable alternative to HiSeq for de novo RNA-Seq assembly.