scholarly journals Rare Variant Phasing and Haplotypic Expression from RNA-Sequencing with phASER

2016 ◽  
Author(s):  
Stephane E. Castel ◽  
Pejman Mohammadi ◽  
Wendy K. Chung ◽  
Yufeng Shen ◽  
Tuuli Lappalainen
Keyword(s):  
Rna Sequencing ◽  
De Novo ◽  
Genomic Analysis ◽  
Population Based ◽  
Rna Seq ◽  
High Confidence ◽  
Population Genetic Analysis ◽  
Haplotype Phasing ◽  
Functional Genomic Analysis ◽  
Expression Studies

Haplotype phasing of genetic variants is important for clinical interpretation of the genome, population genetic analysis, and functional genomic analysis of allelic activity. Here we present phASER, a fast and accurate approach for phasing variants that are overlapped by sequencing reads, including those from RNA-sequencing (RNA-seq), which often span multiple exons due to splicing. This provides 1) dramatically more accurate phasing of rare and de novo variants compared to population-based phasing; 2) phasing of variants in the same gene up to hundreds of kilobases away which cannot be obtained from DNA-sequencing reads; 3) high confidence measures of haplotypic expression, greatly improving power for allelic expression studies.

scholarly journals Variant Phasing and Haplotypic Expression from Single-molecule Long-read Sequencing in Maize

2019 ◽  
Author(s):  
Bo Wang ◽  
Elizabeth Tseng ◽  
Primo Baybayan ◽  
Kevin Eng ◽  
Michael Regulski ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genomic Analysis ◽  
Full Length ◽  
Specific Gene ◽  
Population Genetic Analysis ◽  
Functional Genomic Analysis ◽  
Long Read ◽  
Allele Specific ◽  
Regulatory Effects ◽  
Hybrid Lines

AbstractHaplotype phasing of genetic variants in maize is important for interpretation of the genome, population genetic analysis and functional genomic analysis of allelic activity. Accordingly, accurate methods for phasing the full-length isoforms are essential for functional genomics studies. We performed an isoform-level phasing study in maize, using two inbred lines and their reciprocal crosses, based on the single-molecule full-length cDNA sequencing. To phase and analyze the full-length transcripts between hybrids and parents, we developed a tool called IsoPhase. Using this tool, we validated the majority of SNPs called against matching short-read data and identified cases of allele-specific, gene-level and isoform-level expression. Our results revealed that maize parental lines and hybrid lines exhibit different splicing activities. After phasing 6,907 genes in two reciprocal hybrids using embryo, endosperm and root tissues, we annotated the SNPs and identified large-effect genes. In addition, based on single-molecule sequencing, we identified parent-of-origin isoforms in maize hybrids, distinct novel isoforms in maize parent and hybrid lines, and imprinted genes from different tissues. Finally, we characterized variation in cis- and trans-regulatory effects. Our study provides measures of haplotypic expression that could increase accuracy in studies of allelic expression.

scholarly journals Identification of viruses infecting six plum cultivars in Korea by RNA-sequencing

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9588 ◽  
Author(s):  
Yeonhwa Jo ◽  
Hoseong Choi ◽  
Sen Lian ◽  
Jin Kyong Cho ◽  
Hyosub Chu ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
De Novo ◽  
Reference Database ◽  
Rna Seq ◽  
Rt Pcr ◽  
Virus Identification ◽  
Hop Stunt Viroid ◽  
Chlorotic Leaf ◽  
Peach Trees ◽  
Next Generation Sequencing Ngs

Background Plums are a kind of stone fruit, a category that includes peaches, cherries, apricots, and almonds. In Korea, Japanese plum trees are usually cultivated as they best suit the climate. To date, there have been few studies in Korea on viruses infecting plum trees compared to those infecting peach trees. Methods To identify viruses and viroids infecting plum trees, we collected leaf samples from six different plum cultivars and subjected them to RNA-sequencing (RNA-seq). Six different plum transcriptomes were de novo assembled using the Trinity assembler followed by BLAST searching against a viral reference database. Results We identified hop stunt viroid (HSVd) and six viruses, including apple chlorotic leaf spot virus (ACLSV), little cherry virus-1 (LChV-1), peach virus D (PeVD), peach leaf pitting-associated virus (PLPaV), plum bark necrosis stem pitting-associated virus (PBNSPaV), and prunus necrotic ringspot virus (PNRSV), from six plum cultivars by RNA-seq. RT-PCR confirmed the infection of HSVd and three viruses—ACLSV, PBNSPaV, and PNRSV—in plum trees. However, RT-PCR demonstrated that plum trees in this study were not infected by LChV-1, PeVD, or PLPaV. It is likely that the three viruses LChV-1, PeVD, and PLPaV as identified by RNA-seq were contaminants from other peach libraries caused by index misassignment, which suggests that careful confirmation by other methods should be carried out in next-generation sequencing (NGS)-based virus identification. Taken together, we identified a viroid and three viruses infecting plum trees in Korea.

scholarly journals De NovoAssembly and Characterization ofSophora japonicaTranscriptome Using RNA-seq

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Liucun Zhu ◽  
Ying Zhang ◽  
Wenna Guo ◽  
Xin-Jian Xu ◽  
Qiang Wang
Keyword(s):  
Protein Interactions ◽  
De Novo ◽  
Average Length ◽  
Genomic Analysis ◽  
Interaction Network ◽  
Protein Protein Interactions ◽  
Gene Expressions ◽  
Sophora Japonica ◽  
Protein Protein Interaction ◽  
Functional Genomic Analysis

Sophora japonicaLinn (Chinese Scholar Tree) is a shrub species belonging to the subfamily Faboideae of the pea family Fabaceae. In this study, RNA sequencing ofS. japonicatranscriptome was performed to produce large expression datasets for functional genomic analysis. Approximate 86.1 million high-quality clean reads were generated and assembledde novointo 143010 unique transcripts and 57614 unigenes. The average length of unigenes was 901 bps with an N50 of 545 bps. Four public databases, including the NCBI nonredundant protein (NR), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), and the Cluster of Orthologous Groups (COG), were used to annotate unigenes through NCBI BLAST procedure. A total of 27541 of 57614 unigenes (47.8%) were annotated for gene descriptions, conserved protein domains, or gene ontology. Moreover, an interaction network of unigenes inS. japonicawas predicted based on known protein-protein interactions of putative orthologs of well-studied plant genomes. The transcriptome data ofS. japonicareported here represents first genome-scale investigation of gene expressions in Faboideae plants. We expect that our study will provide a useful resource for further studies on gene expression, genomics, functional genomics, and protein-protein interaction inS. japonica.

Rule-based integration of RNA-Seq analyses tools for identification of novel transcripts

2014 ◽  
Vol 12 (05) ◽  
pp. 1450026 ◽  
Author(s):  
Harshal Inamdar ◽  
Avik Datta ◽  
Sunitha Manjari K ◽  
Rajendra Joshi
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Rna Seq ◽  
Cellular Functions ◽  
High Confidence ◽  
Rule Based ◽  
Profiling Techniques ◽  
Novel Transcripts ◽  
Different Levels ◽  
Sizable Number

Recent evidences suggest that a substantial amount of genome is transcribed more than that was anticipated, giving rise to a large number of unknown or novel transcripts. Identification of novel transcripts can provide key insights into understanding important cellular functions as well as molecular mechanisms underlying complex diseases like cancer. RNA-Seq has emerged as a powerful tool to detect novel transcripts, which previous profiling techniques failed to identify. A number of tools are available for enabling identification of novel transcripts at different levels. Read mappers such as TopHat, MapSplice, and SOAPsplice predict novel junctions, which are the indicators of novel transcripts. Cufflinks assembles novel transcripts based on alignment information and Oases performs de novo construction of transcripts. A common limitation of all these tools is prediction of sizable number of spurious or false positive (FP) novel transcripts. An approach that integrates information from all above sources and simultaneously scrutinizes FPs to correctly identify authentic novel transcripts of high confidence is proposed.

scholarly journals Discovery of Genes Related to Insecticide Resistance in Bactrocera dorsalis by Functional Genomic Analysis of a De Novo Assembled Transcriptome

PLoS ONE ◽  
2012 ◽  
Vol 7 (8) ◽  
pp. e40950 ◽  
Author(s):  
Ju-Chun Hsu ◽  
Ting-Ying Chien ◽  
Chia-Cheng Hu ◽  
Mei-Ju May Chen ◽  
Wen-Jer Wu ◽  
...  
Keyword(s):  
Insecticide Resistance ◽  
De Novo ◽  
Genomic Analysis ◽  
Bactrocera Dorsalis ◽  
Functional Genomic ◽  
Functional Genomic Analysis

Η αλληλεπίδραση των ενδογενών ρετροϊών και της μεταγωγικής οδού των ιντερφερονών με την υπομεθυλιωτική θεραπεία στα Μυελοδυσπλαστικά Σύνδρομα.

2021 ◽  
Author(s):  
Χρυσούλα Κορδελλά
Keyword(s):  
Rna Sequencing ◽  
De Novo ◽  
Rna Seq

Τα μυελοδυσπλαστικά σύνδρομα (ΜΔΣ) και η οξεία μυελογενής λευχαιμία (ΟΜΛ) χαρακτηρίζονται από μη φυσιολογικά πρότυπα μεθυλίωσης του DNA, τα οποία συμβάλλουν στη παθοφυσιολογία της νόσου, με αποτέλεσμα τη μη φυσιολογική επιγενετική καταστολή και διαφοροποίηση των αιμοποιητικών βλαστικών κυττάρων του μυελού των οστών (HSCs). Οι υπομεθυλιωτικοί παράγοντες (ΥΜΠ), οι οποία αντιστρέφουν αυτή την επιγενετική καταστολή, όπως είναι η 5-αζακυτιδίνη (AZA) και η 5-αζα-2'-δεοξυκυτιδίνη (DAC), παραμένουν η κύρια θεραπεία επιλογής για τους ασθενείς με ΜΔΣ υψηλού κινδύνου και ΟΜΛ. Παρόλο αυτά, ο ακριβής μηχανισμός δράσης των ΥΜΠ παραμένει αδιευκρίνιστος, ενώ απουσιάσουν και προγνωστικοί παράγοντες απόκρισης στους ΥΜΠ. Πρόσφατα δεδομένα σε καρκινικές κυτταρικές σειρές δείχνουν ότι η επανενεργοποίηση ενδογενών ρετροϊικών στοιχείων και η επακόλουθη επαγωγή κυτταρικής ενδογενούς αντιϊικής απόκρισης ενδέχεται να αποτελούν το υπόβαθρο της αντικαρκινικής δραστηριότητας των ΥΜΠ. Χρησιμοποιώντας την τεχνική RNA sequencing (RNA-seq), πραγματοποιήθηκε σύγκριση των επιπέδων μεταγραφής των ενδογενών ρετροϊικών στοιχείων σε HSC απομονωμένα από το μυελό των οστών ασθενών με ΜΔΣ και χρόνιας μυελομονοκυτταρική λευχαιμία (CMML) πριν και μετά τη θεραπεία με AZA και σε HSC μυελού των οστών προερχόμενα από υγιείς δότες και ασθενείς με πρωτοπαθή ΟΜΛ. Σε συμφωνία και με προηγούμενες βιβλιογραφικές αναφορές, διαπιστώθηκε ότι η θεραπεία με ΑZA αύξησε τη μεταγραφή των ενδογενών ρετροϊικών στοιχείων. Ωστόσο, η αύξηση αυτή δε συσχετίστηκε με το αποτέλεσμα της θεραπείας με ΑΖΑ, καθώς παρατηρήθηκε τόσο στους αποκρινόμενους όσο καις τους μη αποκρινόμενους ασθενείς. Η παρατήρηση αυτή επιβεβαιώθηκε και με τη τεχνική της de novo συναρμολόγησης του μεταγραφώματος, αποκαλύπτοντας ότι οι, αποκρινόμενοι στην ΑΖΑ, ενδογενείς ρετροϊοί δεν αποτελούν καθοριστικό παράγοντα για το αποτέλεσμα της θεραπείας με ΥΜΠ. Επίσης, παρατηρήθηκε έλλειψη επαγωγής μίας αντιϊικής απόκρισης των ιντερφερονών η οποία να σχετίζεται με την απόκριση στη θεραπεία με ΑΖΑ. Αντίθετα, αποκαλύφθηκε ότι η προθεραπευτική έκφραση και η διαδικασία του εναλλακτικού ματίσματος συγκεκριμένων, αναπτυξιακά ρυθμιζόμενων, γονιδίων μπορεί να αποτελούν προγνωστικούς παράγοντες για την ανταπόκριση των ασθενών με ΜΔΣ και CMML-ΙΙ στη θεραπεία με AZA. Συμπερασματικά, η παρούσα διδακτορική διατριβή αναδεικνύει τη μη συμμετοχή των ενδογενών ρετροϊικών στοιχείων στην αποτελεσματικότητα δράσης των ΥΜΠ. Προσδιορίζει ταυτόχρονα τις αναπτυξιακά ρυθμιζόμενες μεταγραφικές υπογραφές συγκεκριμένων γονιδίων, που είτε κωδικοποιούν την αντίστοιχη πρωτεΐνη είτε όχι, ως συσχετίζομενα με μια ευνοϊκή ανταπόκριση των ασθενών με ΜΔΣ και CMML-ΙΙ στη θεραπεία με AZA, προσφέροντας νέους υποψηφίους δείκτες για περαιτέρω αξιολόγηση.

scholarly journals rnaSPAdes: a de novo transcriptome assembler and its application to RNA-Seq data

GigaScience ◽  
2019 ◽  
Vol 8 (9) ◽  
Author(s):  
Elena Bushmanova ◽  
Dmitry Antipov ◽  
Alla Lapidus ◽  
Andrey D Prjibelski
Keyword(s):  
Rna Sequencing ◽  
De Novo ◽  
Transcriptome Assembly ◽  
The Novel ◽  
Rna Seq ◽  
De Novo Transcriptome ◽  
Weak Points ◽  
Transcriptome Reconstruction ◽  
Evaluation Approaches ◽  
Genome Assembler

Abstract Background The possibility of generating large RNA-sequencing datasets has led to development of various reference-based and de novo transcriptome assemblers with their own strengths and limitations. While reference-based tools are widely used in various transcriptomic studies, their application is limited to the organisms with finished and well-annotated genomes. De novo transcriptome reconstruction from short reads remains an open challenging problem, which is complicated by the varying expression levels across different genes, alternative splicing, and paralogous genes. Results Herein we describe the novel transcriptome assembler rnaSPAdes, which has been developed on top of the SPAdes genome assembler and explores computational parallels between assembly of transcriptomes and single-cell genomes. We also present quality assessment reports for rnaSPAdes assemblies, compare it with modern transcriptome assembly tools using several evaluation approaches on various RNA-sequencing datasets, and briefly highlight strong and weak points of different assemblers. Conclusions Based on the performed comparison between different assembly methods, we infer that it is not possible to detect the absolute leader according to all quality metrics and all used datasets. However, rnaSPAdes typically outperforms other assemblers by such important property as the number of assembled genes and isoforms, and at the same time has higher accuracy statistics on average comparing to the closest competitors.

scholarly journals RNA-Sequencing Based Assessment of Targets, Risk and Long Term Survival for Personalized Treatment of Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1801-1801
Author(s):  
Martina Emde ◽  
Susanne Beck ◽  
Vladimir Benes ◽  
Jerome Moreaux ◽  
Anja Seckinger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Rna Sequencing ◽  
De Novo ◽  
Proliferation Index ◽  
High Dose ◽  
Rna Seq ◽  
Term Survival ◽  
Malignant Plasma Cell

Aim of our study is to implement RNA-sequencing as basis for risk adapted and targeted treatment-strategies in multiple myeloma. We establish assessment of malignant plasma cell proliferation and risk-stratification de novo (HDHRS) by RNA-sequencing, transfer microarray-based risk scores to RNA-sequencing, and report expression of actionable including immune-oncological targets. Methods. Transcriptome profiling was performed on CD138 purified malignant plasma cell samples from previously untreated symptomatic multiple myeloma patients undergoing high-dose melphalan treatment and autologous stem cell transplantation. RNA-sequencing was performed on an Illumina NextSeq500-platform using 5ng of total RNA (n=535), and, in the same cohort, by gene expression profiling using Affymetrix U133 2.0 DNA-microarrays (n=534). iFISH was performed using a ten-probe panel (n=535). Clinical data and survival of patients treated with high-dose therapy and autologous stem cell transplantation were assessed (n=534/533/534 for RNA-seq/GEP/iFISH) with median follow up of 64 months. Data were validated using the independent COMPASS cohort (n=767 pts., median follow up of 31 months). Results. RNA-sequencing is feasible in more than 90% of myeloma patients. It allows assessment of proliferation (RPI) and risk, both de novo (HDHRS) and regarding microarray-based scores (UAMS GEP70-, EMC92-, Rs-score). All scores are significantly predictive for overall and event-free survival in GEP are likewise in RNA-seq with hazard ratio for high risk to low risk group of 3.06, 4.3, 2.84, 3.93 and 3.30 in overall survival of test group. (See as example gene expression based proliferation index / RNA-seq based proliferation index, Fig. 1). The de novo RNA-seq risk stratification (HDHRS) superiorly delineates three significantly different groups of 40%, 38%, and 22% of patients with 5-year survival rates of 84%, 67%, and 32%, respectively (Fig.2). It likewise discerns a group of long term surviving patients with a survival rate of 57% and 49%, at 10 and 12 years, respectively. Targets expressed in normal plasma cells with varying expression height in malignant include BCMA and CD38, those aberrantly expressed CSF1, CD20 and HGF. Mutated actionable transcripts include BRAF V600E/K (target), the proteasome and cereblon (resistance). In conclusion, RNA-seq allows assessment of risk, long term survival and targets in multiple myeloma both in phase III multicenter trials as well as in clinical Routine. Disclosures Moreaux: Diag2Tec: Other: Co-founder of Diag2Tec company.

scholarly journals Multiple Viral Infections Detected in Phytophthora condilina by Total and Small RNA Sequencing

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 620
Author(s):  
Leticia Botella ◽  
Thomas Jung
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Viral Infections ◽  
De Novo ◽  
Phytophthora Species ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Rt Pcr ◽  
Total Rna ◽  
Southern Portugal

Marine oomycetes have recently been shown to be concurrently infected by (−)ssRNA viruses of the order Bunyavirales. In this work, even higher virus variability was found in a single isolate of Phytophthora condilina, a recently described member of Phytophthora phylogenetic Clade 6a, which was isolated from brackish estuarine waters in southern Portugal. Using total and small RNA-seq the full RdRp of 13 different potential novel bunya-like viruses and two complete toti-like viruses were detected. All these viruses were successfully confirmed by reverse transcription polymerase chain reaction (RT-PCR) using total RNA as template, but complementarily one of the toti-like and five of the bunya-like viruses were confirmed when dsRNA was purified for RT-PCR. In our study, total RNA-seq was by far more efficient for de novo assembling of the virus sequencing but small RNA-seq showed higher read numbers for most viruses. Two main populations of small RNAs (21 nts and 25 nts-long) were identified, which were in accordance with other Phytophthora species. To the best of our knowledge, this is the first study using small RNA sequencing to identify viruses in Phytophthora spp.

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