Strand-specific RNA-seq Libraries Research Articles

Abstract 221: Development of a rapid, highly sensitive cell-free RNA NGS assay for the detection of gene fusion and splicing variants in cancers

Abstract Background: Utility of cell-free nucleic acids (cf-NAs) for early detection and surveillance of tumor represents a new path in personalized medicine. Gene fusions, which usually arise from chromosomal rearrangement, play pivotal roles in numerous human malignancies and have been exploited for disease diagnosis and therapeutics. RNA sequencing, which is not influenced by the complication of introns, is considered superior to DNA sequencing for the identification of fusion genes and splicing variants, and has emerged as a critical tool in cancer liquid biopsy. Methods: We developed a novel approach of preparing strand-specific RNA-Seq libraries. The cDNA produced from the cell-free RNA (cfRNA) samples were labelled with unique molecular identifiers (UMI) during library construction. The libraries were enriched using hybrid capture and the sequencing data were analyzed using Predicine in-house analysis pipeline. To assess the performance of the newly developed cfRNA assay, we compared it with a variety of commercial RNA library kits. Results: The new RNA library construction workflow was streamlined, and it reduces the library preparation time from 2 days to 6 hours with less than 1 h of hands-on time. The yield of the library prepared by the new method was higher than those of the commonly used commercial kits on the market. Sequencing analysis and Droplet Digital PCR (ddPCR) measurement both revealed that our assay was more sensitive in detecting gene fusions and splicing variants in cfRNA than other commercial kits. The assay can reliably detect fusion/splicing variants down to the level of <10 copies of input cfRNA with high specificity. Conclusions: We developed a new RNA library preparation method that produced strand-specific libraries from low input quantities of cfRNA in a fast, simple, and robust manner. This RNA-Seq assay has demonstrated superior performance in known fusion detection and novel fusion discovery in cfRNA. Citation Format: fang liu, xiangying sun, jianrong dong, kemin zhou, yong huang, shidong jia, pan du, Binggang xiang, shujun luo. Development of a rapid, highly sensitive cell-free RNA NGS assay for the detection of gene fusion and splicing variants in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 221.

A novel strand-specific RNA-sequencing protocol using dU-adaptor-assembled Tn5.

Strand-specific RNA-seq is a powerful tool for the discovery of novel transcripts, annotation of genomes, and profiling of gene expression levels. Tn5 transposase has been successfully applied in massive-scale sequencing projects; in particular, Tn5 adaptor modification is used in epigenetics, genomic structure, and chromatin visualization. We developed a novel dU-adaptor-assembled Tn5-mediated strand-specific RNA-sequencing protocol and compared this method with the leading dUTP method in terms of experimental procedure and multiple quality metrics of the generated libraries. The results showed that the dU-Tn5 method is easy to operate and generates a strand-specific RNA-seq library of comparable quality considering library complexity, strand-specificity, evenness, and continuity of annotated transcript coverage. We also evaluated the performance of the dU-Tn5 method in identifying nitrogen-responsive protein-coding genes and long non-coding RNAs in soybean roots. The results indicated that ~62-70% of differentially expressed genes detected from conventional libraries were also detected in dU-Tn5 libraries, indicating good agreement of our method with the current standard; moreover, their fold-changes were highly correlated (R>0.9). Thus, our method provides a promising 'do-it-yourself' stranded RNA-seq procedure for gene expression profiling.

471 Integrated psoriasis GWAS and eQTL analysis reveals risk-associated genetic control of TRAF3IP2-AS1 expression in activated CD8 T-cells

We performed an international meta-GWAS of psoriasis, increasing effective sample size by 3.4-fold. Using COJO, we identified 191 independent psoriasis loci at genome-wide (GW) significance (179 outside the MHC). We conducted eQTL analysis of 1,195 strand-specific RNA-seq libraries passing QC (MQ30, ∼25M reads/sample) from 9 FACS-purified immune cell types (CD4/CD8, CLA-/CLA+, and 0h/24 h CD3-CD28-activated T-cells; also resting mDC) from 153 genotyped individuals, yielding 9,091 significant (p<5x10-3) cis-eQTLs mapping to the 179 non-MHC 95% Bayesian credible sets. Here we focus on TRAF3IP2 encoding Act1, an adaptor that transduces signals through IL-17R. COJO found 14 independent risk signals for this region (2 potentially protein-altering and 12 potentially regulatory). The lead protein-coding variant, rs33980500 C/T encoding Act1 D10N, is the top GWAS hit with a posterior probability of 1.00 on unconditional analysis. We have shown that rs33980500-T increases psoriasis risk and promotes Th17 expansion. We identified cis-eQTLs for the lncRNA TRAF3IP2-AS1 in activated CD8 T-cells, but not in activated CD4 or resting T-cells. In contrast, cis-eQTL signals for TRAF3IP2 itself were markedly reduced after CD8 T-cell activation. Moreover, the best cis-eQTL signal for TRAF3IP2-AS1 in activated CD8 T-cells (rs11153301, p=2.9x10-6) is a GW-significant psoriasis hit (p=3.7x10-8) after conditioning on Act1 D10N. The risk allele rs11153301-T results in reduced expression of TRAF3IP2-AS1 in activated CD8 T-cells, and a recent study (J Immunol 206:2353) describes an inhibitory effect of TRAF3IP2-AS1 on Act1 protein level and IL-17 signaling. Notably, the expanded GWAS also implicates a cluster of noncoding variants near IL17RA (p=3.9x10-9), which encodes a key subunit of IL-17R targeted by brodalumab, a highly effective psoriasis treatment. These results suggest that TRAF3IP2-AS1 acts as a brake on psoriasis via its effects on IL-17 signaling in activated CD8 T-cells.

Systematic comparative analysis of strand-specific RNA-seq library preparation methods for low input samples

Despite the recent precipitous decline in the cost of genome sequencing, library preparation for RNA-seq is still laborious and expensive for applications such as high throughput screening. Limited availability of RNA generated by some experimental workflows poses an additional challenge and increases the cost of RNA library preparation. In a search for low cost, automation-compatible RNA library preparation kits that maintain strand specificity and are amenable to low input RNA quantities, we systematically tested two recent commercial technologies—Swift RNA and Swift Rapid RNA, presently offered by Integrated DNA Technologies (IDT) —alongside the Illumina TruSeq stranded mRNA, the de facto standard workflow for bulk transcriptomics. We used the Universal Human Reference RNA (UHRR) (composed of equal quantities of total RNA from 10 human cancer cell lines) to benchmark gene expression in these kits, at input quantities ranging between 10 to 500 ng. We found normalized read counts between all treatment groups to be in high agreement. Compared to the Illumina TruSeq stranded mRNA kit, both Swift RNA library kits offer shorter workflow times enabled by their patented Adaptase technology. We also found the Swift RNA kit to produce the fewest number of differentially expressed genes and pathways directly attributable to input mRNA amount.

RNA-Seq for Bacterial Gene Expression.

RNA sequencing (RNA-seq) has become the preferred method for global quantification of bacterial gene expression. With the continued improvements in sequencing technology and data analysis tools, the most labor-intensive and expensive part of an RNA-seq experiment is the preparation of sequencing libraries, which is also essential for the quality of the data obtained. Here, we present a straightforward and inexpensive basic protocol for preparation of strand-specific RNA-seq libraries from bacterial RNA as well as a computational pipeline for the data analysis of sequencing reads. The protocol is based on the Illumina platform and allows easy multiplexing of samples and the removal of sequencing reads that are PCR duplicates. © 2018 by John Wiley & Sons, Inc.

Landscape of long non-coding RNAs in Trichophyton mentagrophytes-induced rabbit dermatophytosis lesional skin and normal skin.

Emerging evidences suggest that long non-coding RNAs (lncRNAs) play important role in disease development. However, the role of rabbit lncRNAs in the pathogenesis of dermatophytosis remains elusive. The present study aimed to study and characterize lncRNA transcriptome in 8 T. mentagrophytes-induced female rabbit dermatophytosis lesional (TM) and 4 normal saline-infected (NS) skin biopsies using RNAseq. We identified 5883 lncRNAs in 12 strand-specific RNA-seq libraries and found 64 differentially expressed lncRNAs (q<0.05) in TM relative to NS. As in other mammalian counterparts, rabbit lncRNAs were distributed in all chromosomes except the Y chromosome and were generally smaller in size and fewer in exon numbers compared to protein coding genes. Next, co-expression analysis revealed that 107 pairs between 32 DE lncRNAs and 96 protein coding genes showed a highly correlated expression (|r|>0.8). Moreover, miRPara analysis of the lncRNAs revealed 173 lncRNAs with precursor sequences for 9561 probable novel miRNAs. Finally, q-PCR results validated the RNA-seq results with eight randomly selected lncRNAs. To the best of our knowledge, this is the first report on rabbit lncRNAs, and our results highlighted the potential role of lncRNAs in the pathogenesis of dermatophytosis.

(297) - Identification of pain and wound healing-related genes associated with CD163 in macrophages from diabetic patients

Macrophages are pivotal for the induction of pain and tissue repair. In diabetes, macrophages have a persistent pro-inflammatory phenotype that impairs wound healing. We have identified CD163 as a key molecule for the transition from M1 to M2 macrophages. We hypothesize that macrophages from patients with diabetes are dysfunctional due to altered molecular pathways downstream CD163. Using whole-transcriptome analyses and bioinformatics we propose to identify pain and tissue repair related target genes that are modulated by CD163 in diabetic macrophages. Monocytes were isolated from the peripheral blood (healthy and diabetic subjects with or without diabetic peripheral neuropathy or painful diabetic neuropathy, IRB PC-201523), differentiated to macrophages, stimulated with LPS (5 ug/ml, 48 h) and transfected with a CD163 plasmid, or an empty plasmid as control. Strand-specific RNAseq libraries were prepared from 9 samples (3 healthy, 3 diabetic, 3 diabetic neuropathic). Illumina 125 bp paired-end reads were sequenced from each library. Raw read counts were used as input to edgeR for differential gene expression analysis. Up and down-regulated genes with a false discovery rate < 5% were analyzed. We found that the following migration-related genes were of low relevance for diabetic macrophages: FLRT2, DPP4, AXL, and ENPP2. These genes seem to be dependent upon CD163 and relevant for healthy macrophages to induce wound healing. The following migration-related genes were found downregulated and of particular relevance in diabetic macrophages, with no dependence upon CD163: ALOX15B, SDC3, and JAK3, being the latter a pain-related gene. We are currently performing a detailed bioinformatics analysis to expand the implications of other genes in different functional pathways. We have identified potential targets to improve wound healing, which could lead to the development of novel analgesics or disease-modifying therapeutics. Supported by 2016 SC-INBRE Bioinformatics grant, The Rita Allen Foundation and SPARC PC School of Pharmacy grant.

Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing

BackgroundRNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming.MethodsHere, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction.ResultsThis work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments.ConclusionsThese developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.

Abstract 5406: Low-input transcript profiling with enhanced sensitivity using a highly efficient, low-bias and strand-specific RNA-Seq library preparation method

Abstract RNA-seq has become the most popular method for transcriptome analysis and is widely used to study gene expression, detect mutations, fusion transcripts, alternative splicing, and post-transcriptional modifications. It is becoming the method of choice to detect alterations in diseases, especially cancer, to provide insights on the various molecular pathways perturbed by changes in the transcriptome and study their implications. As RNA-seq is being adopted in molecular diagnostics and biomarker identifications, the need for good quality, reproducible library preparation methods using very low amounts of RNA input, or precious clinical samples, is increasing. To address these challenges, we have developed a strand-specific RNA-seq library preparation method that retains information about which strand of DNA is transcribed, from as low as 5 ng total RNA input. Strand specificity is important for correct annotation of genes, identification of antisense transcripts with potential regulatory roles, and accurate determination of gene expression levels in the presence of antisense transcripts. Enhanced sensitivity to detect transcripts with even coverage across their length offers a non-biased approach for accurate quantification of transcript levels. Methods: Enriched poly-A mRNA or ribo-depleted RNA (Universal Human Reference RNA) was used to make libraries with our strand specific library preparation method. Library quality and quantity were analyzed on an Agilent Bioanalyzer, pooled at equimolar ratio and sequenced on Illumina’s Nextseq 500. Paired end reads were mapped to a human reference genome (hg19) using Hisat2 and sequencing metrics were calculated using Picard's RNA-seq Metrics and RSeqC tools. Transcript abundance was measured using Salmon and the Ensembl GRCh38 CDS sequences. Results: Libraries prepared with our streamlined method using inputs that range from 5ng to 1ug show greater than 98% directionality at all input levels. GC content analysis, gene body coverage and gene expression correlation are similar for all inputs tested (5ng to 1ug), even though input amounts vary by over three orders of magnitude. These consistent results are recapitulated with the spiked-in ERCC controls at all inputs. Conclusions: Our library preparation method is streamlined and can be used for a wide range of input RNA without any major modifications to the protocol, making it an easy to follow, convenient method for RNA-seq library preparation. In addition, our method has increased sensitivity and specificity, especially for low-abundance transcripts, reduced PCR duplicates and sequence bias, delivering high quality strand-specific data even for low input RNA. Finally, our method is compatible with both poly A-tail enriched and ribosomal RNA depleted samples, and is amenable to large-scale library construction and automation. Citation Format: Keerthana Krishnan, Erbay Yigit, Mehmet Karaca, Deyra Rodriguez, Bradley Langhorst, Timur Shtatland, Daniela Munafo, Pingfang Liu, Lynne Apone, Vaishnavi Panchapakesa, Karen Duggan, Christine Sumner, Christine Rozzi, Fiona Stewart, Laurie Mazzola, Joanna Bybee, Danielle Rivizzigno, Eileen T. Dimalanta, Theodore B. Davis. Low-input transcript profiling with enhanced sensitivity using a highly efficient, low-bias and strand-specific RNA-Seq library preparation method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5406. doi:10.1158/1538-7445.AM2017-5406

Global transcriptome analysis reveals extensive gene remodeling, alternative splicing and differential transcription profiles in non-seed vascular plant Selaginella moellendorffii

BackgroundSelaginella moellendorffii, a lycophyte, is a model plant to study the early evolution and development of vascular plants. As the first and only sequenced lycophyte to date, the genome of S. moellendorffii revealed many conserved genes and pathways, as well as specialized genes different from flowering plants. Despite the progress made, little is known about long noncoding RNAs (lncRNA) and the alternative splicing (AS) of coding genes in S. moellendorffii. Its coding gene models have not been fully validated with transcriptome data. Furthermore, it remains important to understand whether the regulatory mechanisms similar to flowering plants are used, and how they operate in a non-seed primitive vascular plant.ResultsRNA-sequencing (RNA-seq) was performed for three S. moellendorffii tissues, root, stem, and leaf, by constructing strand-specific RNA-seq libraries from RNA purified using RiboMinus isolation protocol. A total of 176 million reads (44 Gbp) were obtained from three tissue types, and were mapped to S. moellendorffii genome. By comparing with 22,285 existing gene models of S. moellendorffii, we identified 7930 high-confidence novel coding genes (a 35.6% increase), and for the first time reported 4422 lncRNAs in a lycophyte. Further, we refined 2461 (11.0%) of existing gene models, and identified 11,030 AS events (for 5957 coding genes) revealed for the first time for lycophytes. Tissue-specific gene expression with functional implication was analyzed, and 1031, 554, and 269 coding genes, and 174, 39, and 17 lncRNAs were identified in root, stem, and leaf tissues, respectively. The expression of critical genes for vascular development stages, i.e. formation of provascular cells, xylem specification and differentiation, and phloem specification and differentiation, was compared in S. moellendorffii tissues, indicating a less complex regulatory mechanism in lycophytes than in flowering plants. The results were further strengthened by the evolutionary trend of seven transcription factor families related to vascular development, which was observed among four representative species of seed and non-seed vascular plants, and nonvascular land and aquatic plants.ConclusionsThe deep RNA-seq study of S. moellendorffii discovered extensive new gene contents, including novel coding genes, lncRNAs, AS events, and refined gene models. Compared to flowering vascular plants, S. moellendorffii displayed a less complexity in both gene structure, alternative splicing, and regulatory elements of vascular development. The study offered important insight into the evolution of vascular plants, and the regulation mechanism of vascular development in a non-seed plant.

Strand-Specific Transcriptome Sequencing Using SMART Technology.

Next-generation sequencing is empowering a deeper understanding of biology by enabling RNA expression analysis over the entire transcriptome with high sensitivity and a wide dynamic range. One powerful application within this field is stranded RNA sequencing (RNA-seq), which is necessary to distinguish overlapping genes and to conduct comprehensive annotation and quantification of long non-coding RNAs. Commonly used methods for generating strand-specific RNA-seq libraries are often complicated by protocols that require several rounds of enzymatic treatments and clean-up steps, making them time-intensive, insensitive, and unsuitable for processing several samples simultaneously. An additional challenge in the generation of RNA-seq libraries from total RNA involves the high amount of ribosomal RNA (rRNA) in the starting material. This unit presents streamlined workflows for generating strand-specific RNA-seq libraries from 10ng to 1µg total RNA, representing a minimum of 1000 cells, in less than 7hr with minimal carryover rRNA. These methods allow scientists to evaluate the expression of all transcripts, including non-polyadenylated long non-coding RNAs, even in limited biological samples. Combination of the RNase H-based RiboGone rRNA removal system and SMARTer Stranded RNA-seq technology enables depletion of over 95% of rRNA from mammalian samples, and direct production of Illumina-ready libraries that maintain strand-of-origin information. An alternate method for low input of highly degraded samples is also presented. © 2016 by John Wiley & Sons, Inc.

First insights into the nature and evolution of antisense transcription in nematodes.

BackgroundThe development of multicellular organisms is coordinated by various gene regulatory mechanisms that ensure correct spatio-temporal patterns of gene expression. Recently, the role of antisense transcription in gene regulation has moved into focus of research. To characterize genome-wide patterns of antisense transcription and to study their evolutionary conservation, we sequenced a strand-specific RNA-seq library of the nematode Pristionchus pacificus.ResultsWe identified 1112 antisense configurations of which the largest group represents 465 antisense transcripts (ASTs) that are fully embedded in introns of their host genes. We find that most ASTs show homology to protein-coding genes and are overrepresented in proteomic data. Together with the finding, that expression levels of ASTs and host genes are uncorrelated, this indicates that most ASTs in P. pacificus do not represent non-coding RNAs and do not exhibit regulatory functions on their host genes. We studied the evolution of antisense gene pairs across 20 nematode genomes, showing that the majority of pairs is lineage-specific and even the highly conserved vps-4, ddx-27, and sel-2 loci show abundant structural changes including duplications, deletions, intron gains and loss of antisense transcription. In contrast, host genes in general, are remarkably conserved and encode exceptionally long introns leading to unusually large blocks of conserved synteny.ConclusionsOur study has shown that in P. pacificus antisense transcription as such does not define non-coding RNAs but is rather a feature of highly conserved genes with long introns. We hypothesize that the presence of regulatory elements imposes evolutionary constraint on the intron length, but simultaneously, their large size makes them a likely target for translocation of genomic elements including protein-coding genes that eventually end up as ASTs.

Global analysis of the developmental dynamics of Gossypium hirsutum based on strand-specific transcriptome.

Cotton is an economically important crop that provides both natural fiber and by-products such as oil and protein. Its global gene expression could provide insight into the biological processes underlying growth and development, which involve suites of genes expressed with temporal and spatial control by regulatory networks. Generally, the goal for cotton breeding is improvement of the fiber; thus, most previous research has focused on identifying genes specific to the fiber. However, seeds may also play an important role in fiber development. In this study, we constructed and systematically analyzed 21 strand-specific RNA-Seq libraries for Gossypium hirsutum, covering different tissues, organs and development stages, from which approximately 970 million reads were generated to provide a global view of gene expression during cotton development. The organ (tissue)-specific gene expression patterns were investigated, providing further insight into the dynamic programming associated with developmental processes and a way to study the coordination of development between fiber cells and ovules. Series of transcription factors and seed-specific genes have been identified as candidate genes that could elucidate key mechanisms and regulatory networks in nutrient accumulation during ovule development and in fiber development. This study reports comprehensive transcriptome dynamics at various stages of cotton development and will serve as a valuable genome-wide transcriptome resource for initial gene discovery and functional characterization of genes in cotton.

BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction.

Next Generation Sequencing (NGS) is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq) has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing the terminal breathing of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq) reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE) libraries and can easily extend to full transcript coverage shotgun (SHO) type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.

A rapid, simple, and inexpensive method for the preparation of strand-specific RNA-Seq libraries.

High-throughput sequencing of short cDNA tags, or RNA-Seq, has become a staple of genome-wide gene expression studies in plants. RNA-Seq libraries necessarily contain tags that correspond to the mRNA-poly(A) junction, or polyadenylation site, and thus may be mined for data that can help study alternative polyadenylation. This report presents a simple, rapid, and inexpensive method for preparing strand-specific RNA-Seq libraries from varying quantities of total RNA.

Comprehensive analysis of alternative splicing in Digitalis purpurea by strand-specific RNA-Seq.

Digitalis purpurea (D. purpurea) is one of the most important medicinal plants and is well known in the treatment of heart failure because of the cardiac glycosides that are its main active compounds. However, in the absence of strand specific sequencing information, the post-transcriptional mechanism of gene regulation in D. purpurea thus far remains unknown. In this study, a strand-specific RNA-Seq library was constructed and sequenced using Illumina HiSeq platforms to characterize the transcriptome of D. purpurea with a focus on alternative splicing (AS) events and the effect of AS on protein domains. De novo RNA-Seq assembly resulted in 48,475 genes. Based on the assembled transcripts, we reported a list of 3,265 AS genes, including 5,408 AS events in D. purpurea. Interestingly, both glycosyltransferases and monooxygenase, which were involved in the biosynthesis of cardiac glycosides, are regulated by AS. A total of 2,422 AS events occurred in coding regions, and 959 AS events were located in the regions of 882 unique protein domains, which could affect protein function. This D. purpurea transcriptome study substantially increased the expressed sequence resource and presented a better understanding of post-transcriptional regulation to further facilitate the medicinal applications of D. purpurea for human health.

Peregrine

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

Optimizing de novo assembly of short-read RNA-seq data for phylogenomics

BackgroundRNA-seq has shown huge potential for phylogenomic inferences in non-model organisms. However, error, incompleteness, and redundant assembled transcripts for each gene in de novo assembly of short reads cause noise in analyses and a large amount of missing data in the aligned matrix. To address these problems, we compare de novo assemblies of paired end 90 bp RNA-seq reads using Oases, Trinity, Trans-ABySS and SOAPdenovo-Trans to transcripts from genome annotation of the model plant Ricinus communis. By doing so we evaluate strategies for optimizing total gene coverage and minimizing assembly chimeras and redundancy.ResultsWe found that the frequency and structure of chimeras vary dramatically among different software packages. The differences were largely due to the number of trans-self chimeras that contain repeats in the opposite direction. More than half of the total chimeras in Oases and Trinity were trans-self chimeras. Within each package, we found a trade-off between maximizing reference coverage and minimizing redundancy and chimera rate. In order to reduce redundancy, we investigated three methods: 1) using cap3 and CD-HIT-EST to combine highly similar transcripts, 2) only retaining the transcript with the highest read coverage, or removing the transcript with the lowest read coverage for each subcomponent in Trinity, and 3) filtering Oases single k-mer assemblies by number of transcripts per locus and relative transcript length, and then finding the transcript with the highest read coverage. We then utilized results from blastx against model protein sequences to effectively remove trans chimeras. After optimization, seven assembly strategies among all four packages successfully assembled 42.9–47.1% of reference genes to more than 200 bp, with a chimera rate of 0.92–2.21%, and on average 1.8–3.1 transcripts per reference gene assembled.ConclusionsWith rapidly improving sequencing and assembly tools, our study provides a framework to benchmark and optimize performance before choosing tools or parameter combinations for analyzing short-read RNA-seq data. Our study demonstrates that choice of assembly package, k-mer sizes, post-assembly redundancy-reduction and chimera cleanup, and strand-specific RNA-seq library preparation and assembly dramatically improves gene coverage by non-redundant and non-chimeric transcripts that are optimized for downstream phylogenomic analyses.

Comparative analysis of strand-specific RNA sequencing approaches

Background Standard RNA sequencing approaches generally require double-stranded cDNA synthesis, which erases RNA strand information. Synthesis of a randomly primed double-stranded cDNA followed by addition of adaptors for next-generation sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Different strand-specific RNA-seq approaches have been developed to preserve information about strand polarity with different level of performances. Material and methods Using Illumina Deep Sequencing Technology, this work investigates the performance of two different directional RNA-Seq (strand-specific RNA-seq) strategies. One is based on direct ligation of adaptors to the RNA ends and the other is based on the labeling and excision of the second strand cDNA. The RNA-seq workflows present in this work have been improved over current more laborious RNA-seq methods. Their low RNA input and streamlined workflows make them compatible with high throughput and automation. We also analyze the effect of different RNA fragmentation methods (divalent cations plus heat versus enzymatic fragmentation). Results We will provide a comparative full data analysis of different strand-specific RNA methods (library performance, complexity, continuity of gene coverage, strand specificity, rRNA background). Conclusions Our results show improved methods for high-quality strand-specific RNA-seq library construction amenable to large-scale library construction and automation.

A simple strand-specific RNA-Seq library preparation protocol combining the Illumina TruSeq RNA and the dUTP methods

Preserving the original RNA orientation information in RNA-Sequencing (RNA-Seq) experiment is essential to the analysis and understanding of the complexity of mammalian transcriptomes. We describe herein a simple, robust, and time-effective protocol for generating strand-specific RNA-seq libraries suited for the Illumina sequencing platform. We modified the Illumina TruSeq RNA sample preparation by implementing the strand specificity feature using the dUTP method. This protocol uses low amounts of starting material and allows a fast processing within two days. It can be easily implemented and requires only few additional reagents to the original Illumina kit.