Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA–protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult.We excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step.We successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well.We present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.The online version of this article (doi:10.1186/s12867-017-0099-7) contains supplementary material, which is available to authorized users. Formalin-fixed paraffin-embedded (FFPE) tissue samples constitute a vast and valuable resource of patient material that can potentially be used for biomedical research. In most cases such specimen also entail large amounts of clinically relevant data, such as clinical history, further laboratory findings, follow-up data, and much more. To date however, the extraction of macromolecules in general, and RNA in particular, from FFPE tissues has proved challenging due to chemical crosslinking of RNA with proteins and RNA fragmentation, both of which heavily decrease RNA quantity that can be extracted from such tissues, and also severely impact on RNA quality for downstream analysis e.g. –4)..4]).Laser-capture microdissection (LCM) is a technique that allows the specific isolation of defined areas, such as particular subpopulations of cells, from within a tissue section by direct microscopic visualization . This apDue to all these facts, to date most LCM analyses geared towards RNA expression analysis have used fresh-frozen rather than FFPE tissue sections . HoweverWe have recently established a procedure to extract cancer-associated stroma (CAS) from archival canine FFPE breast cancer tissue by LCM using reverse-transcription quantitative PCR (RT-qPCR) . During http://support.moleculardevices.com). To visualise the areas of interest, tissue sections were stained with Cresyl Fast Violet according to [Thirteen dog mammary carcinoma samples were obtained from the Institute of Veterinary Pathology of the Vetsuisse Faculty Zürich Table . All samrding to  with sli® CapSure® Macro LCM Caps (Life Technologies) were used. Highly enriched populations of normal or tumour-associated stroma from the specimen were identified and isolated according to the manufacturer’s protocol. Normal stroma samples were isolated from the same slides, from regions specified by a pathologist (AM) that presented no obvious alterations or at least 2 mm away from the tumour [® Safe-Lock Tubes) and placed on ice until proceeding with mRNA isolation. 1–2 caps were used per sample.Before microdissection, identification of tumour stroma in samples was performed by a pathologist . Criteria for stroma: fibroblastic cells, endothelial cells and pericytes of small vessels, only single inflammatory cells to avoid areas with heavy inflammation, no adipocytes. For Microdissection the ArcturusXT™ Laser Capture Microdissection System (Thermo Scientific) and Arcturuse tumour . Isolatihttps://tools.thermofisher.com). To elute the RNA from the column, RNase-free water was used to avoid the effects of elution buffer on downstream applications. The eluate was aliquoted before analysis and stored at −80 °C. RNA abundance and quality was analysed using the 4200 or 2200 Tape Station Software using the High Sensitivity RNA ScreenTape kit (Agilent Technologies), according to the manufacturer’s protocol.Extraction of mRNA was performed immediately after microdissection using the Recover All™ Total Nucleic Acid Isolation Kit for FFPE (Ambion™) according to the manufacturer’s protocol with the following small adjustments. As long exposure to xylene has been shown to be detrimental to mRNA integrity , and dep® truXTRAC FFPE RNA kit according to the manufacturer’s protocol with following adjustments. A sterile blade was used to peel off the thermoplastic film from the LCM cap and transfer the tissue isolated by LCM into glass vials for sonication. Sonication of samples was performed with the E220 focused ultrasonicator (Covaris®). After reverse crosslinking at 80 °C, the soluble fraction was transferred into clean eppendorf tubes and DNAse treated without prior centrifugation, as due to the absence of paraffin, the samples did not contain any solids that could be precipitated. RNA was eluted from the spin columns using 30 µl of elution buffer prewarmed to 70 °C to increase RNA yield. A second elution into fresh collection tubes was performed using 20–30 µl prewarmed elution buffer using the identical elution protocol. The eluate was aliquoted before analysis and stored at −80 °C. RNA abundance and quality was analysed using the 4200 or 2200 Tape Station Software using the High Sensitivity RNA ScreenTape kit (Agilent Technologies), according to the manufacturer’s protocol.Extraction of RNA was performed immediately after microdissection using the Covaris® PreAmp Master Mix (2×) (Applied Biosystems™). This step was necessary due to the very low concentrations of RNA extracted using the old protocol, but can be omitted using the new protocol that yields much higher RNA amounts and concentration. For comparability reasons it was performed identically in parallel for all samples. The preamplification was performed according to the manufacturer’s protocol using 14 PCR cycles. RT-qPCR was performed using KAPA PROBE FAST qPCR Kit Master Mix (2×) Universal reagents (Kapa Biosystems), with 2.5 µl cDNA per reaction, in a total volume of 10 µl. RT-qPCR were run in duplicates on the CFX384 Touch™ Real-Time PCR detection system (BioRad). Details regarding primers can be found in [Reverse transcription was performed using the iScript™ cDNA Synthesis Kit (BioRad) according to the manufacturer’s protocol, using a maximum of 10 µl of RNA per reaction. Due to the limiting concentration extractions using the old method, RNA inputs per reaction ranged between 0.5 and 2.3 ng of total RNA per sample. This kit allows generation of cDNA with combination of oligo(dT) and random hexamer primers using low RNA inputs and is optimized for fragments below 1 kb of length. To increase the number of RT-qPCR analyses that could be performed, cDNA was preamplified using the TaqManfound in .http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were trimmed with Trimmomatic [RNA library preparation and depletion of ribosomal RNA was performed using the SMARTer Stranded Total RNA-seq Kit—Pico Input Mammalian from Clontech/Takara Bio USA according to the manufacturer’s protocol with 2 ng input RNA for the ‘old’ extraction protocol, and 10 ng input RNA for the ‘new’ extraction protocol. Single-read sequencing 125 bp) was run on the Illumina HiSeq 2500 using the HiSeq SBS Kit v4 and HiSeq cluster kits v4 according to standard protocols used at the Functional Genomics Centre Zurich (FGCZ). Resulting NGS reads were quality-checked with FastQC  was used.For all statistical analysis and graphical display the program GraphPad Prism  digestion step to lyse the tissue and liberate RNA Fig. a, usuallIn our experiments, the major bottleneck of RNA extraction using the classical approach seemed to derive from the inefficiency of Proteinase K to completely digest the very fibrous tissue  that had been isolated by LCM. Despite trying marked increases in digestion time , weTo overcome this problem, we decided to test a novel approach for isolation of RNA from FFPE tissue, which relies on tissue disruption using focused ultrasonication prior to the proteinase K treatment step Fig. b. The maTo analyse the performance of the ‘new’ RNA isolation protocol, we isolated comparable amounts of normal stroma and CAS by LCM from the identical FFPE tissue blocks that had been used for our previous study, in which RNA had been isolated by the conventional ‘old’ method  and Tabl. In ordeComparing the results from both extraction methods, we observed a vast increase in RNA yield that could be isolated from the LCM specimen using the new isolation method Fig. b. In theTo compare the performance of RNA extracted using the two methods by RT-qPCR, we randomly picked several of our samples, reverse transcribed identical amounts of RNA extracted using both methods into cDNA in parallel, and analysed expression of the two housekeeping genes GAPDH and B2M by RT-qPCR. RNA isolated using the new method performed in general better yielding lower mean Cq values in RT-qPCR than RNA deriving from the old isolation procedure Table . On averp = 0.5936). Furthermore, we investigated how many mapped reads were informative in respect to exonic regions of the genome. The fraction of reads mapping to exonic regions was between 14 and 17.07% (Fig. To compare the performance of the extracted RNA from both protocols using RNAseq, we checked the overall mapping rate for all generated reads. We observed that, regardless of the isolation protocol used, 49.45–79.51% of the reads were mappable to the dog genome (Fig. Concluding, the vastly increased yield combined by its compatibility with qPCR and NGS makes the new extraction method a preferential choice for extraction of RNA from limiting amounts of FFPE clinical specimen.FFPE tissue archives constitute a vast treasury of valuable samples for biomedical research. Thus far however, the extraction of RNA from FFPE tissues has proved challenging due to chemical crosslinking of RNA with proteins as well as RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. When, additionally to these difficulties, sample size become very small, e.g. due to the need to use LCM to isolate specific subpopulations of cells, it has been very difficult to recover enough RNA that is amenable for proper analysis using RT-qPCR or NGS.Encountering this problem, we found that commonly used RNA isolation protocols were mostly inefficient due to insufficient initial steps of tissue disruption using proteinase K, which most of the time left behind still macroscopically visible pieces of tissue. To overcome this bottleneck, we successfully adapted an isolation protocol for paraffinised FFPE samples that uses focused ultrasonication for efficient tissue disruption to our deparaffinised, stained and laser-capture-microdissected samples.Using this approach, we could show that total RNA yields could be increased 8–12-fold compared to the commonly used proteinase K-based tissue disruption techniques Fig. . More imUsing this optimised RNA extraction protocol, analysis of limiting samples derived from LCM extracted deparaffinised and stained FFPE tissue samples becomes technically feasible. We thus envisage that the application of this protocol can have a tremendous positive impact on the feasibility of RNA expression studies on archival patient-derived samples. The possibility to study gene expression signatures in specific sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation. Importantly, this approach is perfectly translatable to human tissue specimen and different pathologies, and we hope to enable a variety of studies to come to a successful completion using our RNA extraction and NGS-protocols.The sequence data of this study have deposited in the European Nucleotide Archive with the primary Accession Code PRJEB20761.


Lobelia chinensis herbal product has been challenging, since current detection technologies are not effective due to their own limits. High Resolution Melting (HRM) has emerged as a powerful new technology for clinical diagnosis, research in the food industry and in plant molecular biology, and this method has already highlighted the complexity of species identification. In this study, we developed a method of species specific detection of L. chinensis using HRM analysis combined with internal transcribed spacer 2. We then applied this method to commercial products purporting to contain L. chinensis. Our results demonstrated that HRM can differentiate L. chinensis from six common adulterants. HRM was proven to be a fast and accurate technique for testing the authenticity of L. chinensis in herbal products. Based on these results, a HRM approach for herbal authentication is provided.There is an unmet need for herbal medicine identification using a fast, sensitive, and easy-to-use method that does not require complex infrastructure and well-trained technicians. For instance, the detection of adulterants in  Lobelia chinensis Lour. belongs to the family Campanulaceae, and is distributed widely in East Asian countries including China, Korea, and Japan  DNA barcoding coupled with HRM analysis is a very accurate method for the authentication of Scutellaria barbata D. Don., Scutellaria indica L., Oldenlandia diffusa (Willd.) Roxb. Hort. Beng. and Mazus pumilus (Burm. f.) Steenis and Pratia nummularia A. Br. et Aschers. as major adulterants of L. chinensis herbal products  following the manufacturer’s protocol. Final concentrations of all genomic DNA samples were adjusted to 50 ng/μL. The DNA was stored at -20°C for further use.A conserved region of the ITS2 region of nuclear ribosomal DNA was chosen for the identification assay because of its low intraspecific variation and the fact that it has sufficient variability to distinguish between even closely related species . ITS2 unF) over temperature (T) (dF/dT) curve displays the melting temperature (Tm), and the normalized raw curve depicts the decreasing fluorescence against increasing temperature. Genotypes of test samples were identified by selecting a representative sample for each species. Based on a confidence threshold of 90%, a confidence percentage for each genotype was calculated  to identify unknown adulterants by BLAST.The tested products that presented unmatched melting curves were directly sequenced using a 3730XL sequencer . Proofreading and contig assembly of sequencing peak diagrams were performed using CodonCode Aligner V 3.7.1 . All sequences were submitted to TCM Barcode .In order to develop a fast method combining DNA barcoding and HRM analysis for discrimination of L. chinensis as a genotype and using its melting curve as the baseline, by subtracting the melting curves of the other species, we were able generate difference data to estimate the similarity between the melt profiles of the ITS2 amplicons of L. chinensis and the five adulterant species tested in this study. Furthermore, genotype confidence percentages (GCPs) were calculated, with a cut-off value of 90% used to assign a genotype for each barcode region. With this approach, all samples were successfully genotyped, and the five adulterant species were specifically and confidently identified. Visualization and separation of variant melting curves is shown in Figure 1B. Furthermore, the HRM method permits use melting curves to discriminate between the three different haplotypes of L. chinensis, which differ by C/T base transposition at position 26 and A/T base deletion at positions 29 and 30; these results were validated by sequencing of amplified products. Thus, we conclude that HRM analysis of universal ITS2 amplicons is a powerful tool for the identification of L. chinensis and its adulterants.In order to better visualize small differences between individual melting curves, HRM software applications allow calculation of a difference plot. By assigning L. chinensis,” were available to patients and consumers, and contained genomic DNA of sufficient quality and quantity for sequence amplification. Normalized HRM curves for ITS2 amplicons from these species in 20 commercial “L. chinensis” herbal products are shown in Figure 2. The melt analysis of the products of HRM-PCR amplification from L. chinensis gDNA showed three unique melting curves, due to intraspecific variation in the ITS2 sequence. However, out of 20 commercial samples, only 14 products contained L. chinensis as indicated; despite heavy processing, these samples produced melting curves and inflection shapes identical to those produced by ITS2 amplification of pure L. chinensis genomic DNA. The respective melting curve analyses revealed the amplification of similar products since they exhibited the same melting curve profile.After confirmation that all adulterant species can be identified by HRM analysis we used the same protocol to test for adulterants present in commercial herbal products. A total of 20 test samples were purchased from medicine markets, drug stores, and hospitals. All of samples were labeled as “O. diffusa, and one sample was consistent with S. barbata. In addition, the other three samples likely did not contain any L. chinensis whatsoever, but rather contained some other species. In order to find substituted species in these products, DNA barcoding is one of the best solutions. As can be seen from our previous works, DNA barcoding was performed to detect the adulteration and substitution of herbal drugs and found that herbal products sold on the markets were contaminated or substituted with alternative (often cheaper-to-produce) plant species that are not listed on ingredient labels. The ITS2 amplicons of these species did not correspond to the melt profiles of L. chinensis or any of the six tested adulterants; therefore, DNA sequencing of ITS2 regions was performed to identify species in these products. The BLAST result showed that these products have a sequences similar to those of Polygala tenuifolia Willd., Artemisia annua Linn., and Digitaria sanguinalis (L.) Scop., respectively. This finding provides evidence that aerial part without obvious characteristic from annual herbs are easily adulterated or substituted. Therefore, the plant substitution in L. chinensis herbal products sold in the Chinese local market, and may be a serious issue for consumers.However, the melting curves of the other six products generated different melting profiles. Two tested samples were similar to psbA-trnH, rbcL, matK, rpoC, trnL, etc.) or their combinations. Fourth, model melt curves should be constructed, both for the authentic herb and for common adulterants. Finally, the HRM assay system can be used to identify commercial herbal products. PCR product with one barcode can be used to sequence and blast for characterize herbal identity.High Resolution Melting analysis has become a reliable and highly useful molecular technique in many fields, including infectious disease identification, food contaminant screening, and genotyping . HRM tecL. chinensis have been contaminated with other low-cost species. These commercial herbal products, sold in local markets, occur in many different forms which lost obvious character for authentication. This variety makes accurate identification of the constituent species difficult. HRM screening permits discrimination of single nucleotide differences in DNA. In this study, HRM analysis was performed on amplicons from the ITS2 DNA barcoding locus, which has highly conserved nucleotide sequences at the species level. We used an HRM protocol to develop a single and fast test to determine the purity of L. chinensis herbal products, as well as to identify adulterant species. In previous studies, DNA barcoding has proven effective for detecting pure substitution in herbal medicines (At present, many herbal plant products distributed in Chinese local markets lack verification of the authenticity of their contents. Furthermore, as mentioned above, extracts containing edicines . Howeveredicines . Anotheredicines .L. chinensis herbal products currently on the market. When amplified using universal barcoding ITS2 primers, DNA extracted from all herbal specimen tested yielded specific amplification products. L. chinensis and its adulterants were easily distinguished by examining the HRM curves for these amplicons. Among 20 commercial herbal products sold in drug stores and markets in China as “L. chinensis,” we used HRM curves to confirm that while 14 tested samples were uncontaminated, six products were found to contain adulterant plant species. HRM technology was shown to be a fast and accurate closed tube post-PCR method which permits the identification of L. chinensis and its adulterants, thereby guaranteeing clinical drug safety and ensuring the vital interests of patients.This study was the first to use DNA barcoding coupled with HRM analysis to detect the species composition of WS, SY, and JL finished experiments and data analyses. CX, LW, LX, YS, and BD contributed to collect materials. WM and SC designed this experiment.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized.Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25’s endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP.Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity.The online version of this article (doi:10.1186/s12915-017-0444-9) contains supplementary material, which is available to authorized users. More than 300 novel RNA-binding proteins have been identified in HeLa , HEK293 Although TRIM25 has been generally well characterized, the functional importance of its RNA-binding activity remains unclear. Our recent study provided the first clues, showing that the binding of TRIM25 to pre-let-7 leads to its more efficient Lin28a-mediated uridylation . This fiHerein, using data from a proteome-wide analysis of RNA-binding domains, RNA pull-downs, and an electrophoretic mobility shift assay (EMSA), we show that the PRY/SPRY domain of TRIM25 harbors its RNA-binding activity. Furthermore, TRIM25 CLIP-seq and quantitative SILAC-based co-immunoprecipitation assays reveal a comprehensive overview of interacting RNAs and proteins, respectively. Our CLIP-seq data identifies hundreds of coding and non-coding RNAs bound by TRIM25. We show that TRIM25 binds a distinct set of RNAs and prefers to bind GC-rich sequences. We also demonstrate that TRIM25 associates with many RNA-binding proteins, thus suggesting it could play a role in regulating RNA metabolism. Crucially, we present evidence that the binding of TRIM25 to RNA is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. Finally, by showing that the PRY/SPRY regions of other TRIM proteins rescue TRIM25’s RNA-binding activity, we propose that TRIM25 represents a novel group of E3 ligases with RNA-binding activity that use PRY/SPRY as a novel RNA-binding domain. These results provide original insights into the RNA-mediated activities of RNA-binding E3 ubiquitin ligases.E. coli  in TRIM25 KO cells and performed anti-T7 co-immunoprecipitation (co-IP) assays Fig. . Our resTo find TRIM25-interacting proteins, we performed co-IPs of T7-tagged TRIM25 in HeLa cells coupled with quantitative SILAC mass spectrometry. In brief, wild-type T7-tagged TRIM25 was expressed in HeLa cells, and the co-IP was performed using protein A agarose beads coupled with the anti-T7 antibody Fig. . The conNext, to see which of these protein–protein interactions are mediated by RNA, we performed the same co-IP assays using T7-tagged TRIM25ΔRBD Fig.  or T7-taWe set out to find out if TRIM25 KO results in global perturbation of protein levels. To do so, we used SILAC mass spectrometry on the wild-type and TRIM25 KO HeLa cells. Our results showed that the levels of only a few proteins were affected more than two-fold . After UV crosslinking and immunoprecipitation, we observed a specific RNA-protein complex   Fig. . When we16) Fig. . Further16) Fig. . TRIM25 16) Fig.  and theyTRIM25 mRNA was much higher than that on other substrates . Importantly, T7-TRIM25ΔRBD failed to precipitate any RNAs apart from TRIM25 mRNA itself  between protein modification and RNA pull-down efficiencies  Fig. . Moreoveies Fig. . AltogetTo test if RNA was necessary for efficient ubiquitination of ZAP, a physiologically important TRIM25 target, we performed in vitro ubiquitination assays of ZAP (ZAP short) with the TRIM25 constructs described earlier Fig. . IncubatTRIM25 mRNA in particular, can augment its autoubiquitination. Finally, we demonstrated that RNA is an important constituent of TRIM25’s ubiquitin ligase activity towards itself and its physiologically relevant protein target ZAP. Recently, ZAP was shown to suppress HIV-1 replication by binding CG-rich regions in viral RNAs [TRIM25 E3 ubiquitin ligase is a new RNA-binding protein with a role in Lin28a-mediated uridylation of pre-let-7 transcripts . Here, wral RNAs . We propOur results carry very important functional implications given that the 3′-UTRs are platforms for regulatory RNA-binding proteins and that the lengths of the 3′-UTRs associated with specific mRNAs are variable. For example, cancer cells have mRNAs with much shorter 3′-UTRs, which exhibit increased stability and produce more proteins in part through the loss of microRNA-mediated repression . AdditioPreviously, the CC domain of TRIM25 has been implicated in binding to RNA . TRIM25 Importantly, there are more than 80 TRIM proteins in the human genome , dividedTRIM25 is involved in normal development and disease . For exaIn summary, we have presented the transcriptome and proteome-wide interactomes of a novel RNA-binding protein, TRIM25. We showed that the PRY/SPRY domain is responsible for the ability of TRIM25 to bind RNA. Finally, we have provided evidence that RNA is an important component in TRIM25’s ubiquitin ligase activity. A recent genome-wide bioinformatics screen of the human proteome identified more than 2600 proteins with RNA-binding capacity . Unravel3 counts per minute, ~0.1 pmol) and the indicated amounts of proteins produced in E. coli. Binding reactions were performed in 16-μL reactions containing gel exclusion buffer (4.8 mM Tris (pH 8), 144 mM NaCl, 0.96 mM DTT) supplemented with 3 mM MgCl2, 0.5 mM ATP, and 37.5 mM creatine phosphate. Reactions were incubated at 4 °C for 1 h, followed by electrophoresis on a 6% w/v non-denaturing polyacrylamide gel. The signal was recorded with radiographic film or as exposed to a phosphoimaging screen and scanned on a FLA-5100 scanner (Fujifilm).EMSAs were performed with an end-labeled pre-let-7a-1 probe . After the final wash, the proteins associated with the beads were analyzed by western blotting.RNA pull-down assays were performed as previously described , with slP value cutoff = 0.001; false discovery rate Q-value cutoff = 0.1; Jaccard coefficient = 0.25). Enriched protein sets are represented as nodes (red circles) connected by edges (green lines), with the sizes of nodes and edges representing the number of proteins in gene sets and the amount of overlap.Extracts prepared from HeLa cells transfected with plasmids expressing T7-tagged or EGFP-tagged proteins were incubated with protein A agarose with the anti-T7 antibody. The bound proteins were separated on a 4–12% SDS polyacrylamide gel and analyzed by western blotting. As indicated, some samples were treated with RNases A and T1 prior to loading on the gel. For mass spectrometry, the T7-bound beads were incubated with HeLa extracts grown in Light or Heavy R6K4 (13C-labeled arginine and 2D-labeled lysine) isotopes (Dundee Cell Products LM014/16). Next, the bound proteins were run on a 4–12% SDS polyacrylamide gel followed by in-gel digestion as described previously . Gene OnThe protocol was adapted from Moore et al.  using adThe RIP experiments were performed as in the CLIP-seq immunoprecipitations, except for cross-linking and RNase treatment. The bound RNAs were isolated by Trizol extraction, and RNAs were identified by qRT-PCR, using the SuperScript III Platinum SYBR Green One-Step qRT–PCR Kit (Life Technologies) following the manufacturer’s instructions on a Roche 480 LightCycler.Immunoprecipitation was performed as described above. Following the final wash, proteins bound to beads were incubated with Ube1, His-Ube2D3, and ubiquitin (UBPBio), where 1 mg/mL of Ube1, 2 mg/mL of His-Ube2D3, and 50 mg/mL of ubiquitin were used for TRIM25 autoubiquitination and 5 mg/mL of Ube1, 10 mg/mL of His-Ube2D3, and 250 mg/mL of ubiquitin were used for ZAP ubiquitination. The reactions were carried out at 37 °C for 1 hour or as otherwise indicated and were stopped by addition of loading buffer and reducing agent. The protein ubiquitination was analyzed by western blot with anti-TRIM25 or anti-ZAP antibodies.Total protein samples were resolved by standard NuPAGE SDS-PAGE electrophoresis with MOPS running buffer (Life Technologies) and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4 °C with 1:10 western blocking reagent (Roche) in Tris-buffered saline buffer with 0.1% Tween-20 (TBST). The following day, the membrane was incubated for 1 hour at room temperature with the primary antibody solution in 1:20 western blocking reagent diluted in TBST. Antibodies used included rabbit polyclonal anti-TRIM25 , rabbit polyclonal anti-DHX9 , mouse monoclonal anti-tubulin , mouse monoclonal anti-T7 tag HRP conjugate , mouse monoclonal anti-HA-probe HRP conjugate , rabbit monoclonal anti-DDX5 , and rabbit polyclonal anti-ZAP (ZC3HAV1) . After washing in TBST, the blots were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase and detected with the SuperSignal West Pico detection reagent (Thermo Scientific) or WesternSure ECL substrate (Licor). The membranes were stripped using the ReBlot Plus Strong Antibody Stripping Solution (Chemicon) equilibrated in water, blocked in 1:10 western blocking solution in TBST, and re-probed as described above.13C-labeled arginine and 2D-labeled lysine) isotopes (Dundee Cell Products LM014/16). Fragments encoding proteins of interest were cloned into the pCG T7 plasmid and transiently expressed in HeLa cells using Lipofectamine 2000 (Invitrogen). For RNA stability assays, 10 μg/mL of actinomycin D (Sigma) was added to cells and RNA extracted at the time points indicated using Tri Reagent (Sigma). Levels of RNAs of interest were determined by qRT-PCR using SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Thermo-Fisher). A total of 200 ng of RNA were loaded per well for RNAs of interest and these levels were normalized to 18S Ribosomal RNA, for which 1 ng of total RNA was loaded per well (1 in 200 dilution).HeLa cells (American Type Culture Collection) were maintained in standard Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies), supplemented with 10% fetal bovine serum (Life Technologies). For SILAC mass spectrometry, cells were cultured in DMEM supplemented with Light or Heavy R6K4 (600 reached 0.6. The bacteria were induced by 0.5 mM IPTG and incubated for 24 hours at 30 °C with agitation. Pellets were lysed with a cell disruptor in lysis buffer (20 mM NaPPi (pH 7.4), 500 mM NaCl, 5 mM MgCl2, protease inhibitor, DNase, and RNase or benzonase), and lysates were loaded onto an IMAC HiTrap 1-mL FF column in binding buffer (20 mM NaPPi (pH 7.4), 500 mM NaCl, 10 mM imidazole) and eluted with increasing amounts of elution buffer (20 mM NaPPi (pH 7.4), 500 mM NaCl, 500 mM imidazole). Protein fractions were further purified with a gel exclusion column (Superdex 200 30/100 GL or HiLoad Superdex 200 16/60) in gel exclusion buffer (10 mM Tris (pH 8.0), 300 mM NaCl, 2 mM DTT). Selected fractions were pooled and concentrated using Vivaspin 5 30,000 MWCO (Sartorius Stedim UK Ltd.). His-Lin28s was produced and purified as previously specified [The full-length human TRIM25 and RBD mutant were cloned into pET30a, which was transformed into BL21 gold cells. Bacteria was grown in Superbroth in the presence of kanamycin at 37 °C until the ODpecified .Thermal denaturation assays were performed on a Bio-Rad IQ5 ICycler. Recombinant proteins (~4 μM) were mixed with Sypro orange (×5) in a 50-μL reaction. Fluorescent readings were taken every 30 seconds between 20 °C and 70 °C. The melting temperature was determined from the maximum of the first derivative of the melting curve, and the mean melting temperature of three replicates was calculated for each protein.Mass measurements were performed on a Viscotek MALS-20 and VE3580 RI detector attached to an ÄKTA 10 Purifier with microflow components. Protein samples were run at approximately 0.75 mg/mL on a Superdex200 Increase 10/300 GL column, running at 1.0 mL/min in gel exclusion buffer (with 0.5 mM TCEP instead of DTT) with 100-μL injections.HeLa cells were transfected with 200 ng of GeneArt CRISPR Nuclease mRNA (Thermo-Fisher) in addition to 50 ng of each of two sgRNAs (TRIM25 Left sgRNA – CCACGTTGCACAGCACCGTGTTC and TRIM25 Right sgRNA CTGCGGTCGCGCCTGGTAGACGG) targeting sequences in exon 1 of TRIM25. After 24 hours, cells were seeded to a 96-well plate such that there was on average 1 cell per well, and the cells were grown until colonies were visible to the naked eye. Next, cells were split into two 96-well plates, one of which was used for a dot blot analysis. For the dot blot analysis, cells were washed twice in PBS prior to the addition of 30 μL of Passive Lysis Buffer (Promega) per well and incubation for 15 minutes at room temperature. Two microliters of protein from each well was spotted directly onto a nitrocellulose membrane, followed by western blotting, as described above. Selected clones were seeded from the second 96-well plate into 6-well plates, grown, and TRIM25 levels were validated by standard western blotting.L5Ba: 5′-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrArGrArGrCrN-OH-3′. L5Bb: 5′-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrGrUrGrArGrCrN-OH-3′. L5Bc: 5′-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrCrArCrUrArGrCrN-OH-3′. L5Bd: 5′-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrUrCrUrCrUrArGrCrN-OH-3′. 3′ adapter: 5′App-NAGATCGGAAGAGCACACGTCTG-ddC 3′. RT Oligo: 5′-CAGACGTGTGCTCTTCCGATCT-3′.P5_Forward: 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′. P3_barcode_Reverse: BC1: 5′CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′ (has indexing barcode IDX1). BC2: 5′CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′ (has indexing barcode IDX2).


We find that SMAD3 binds poorly to single- and double-stranded RNA, regardless of sequence. However, SMAD3 binds RNA with large internal loops or bulges with high apparent affinity. This apparent affinity matches that for its canonical dsDNA ligand, suggesting a biological role for RNA binding by SMAD3.Several members of the SMAD family of transcription factors have been reported to bind RNA in addition to their canonical double-stranded DNA (dsDNA) ligand. RNA binding by SMAD has the potential to affect numerous cellular functions that involve RNA. However, the affinity and specificity of this RNA binding activity has not been well characterized, which limits the ability to validate and extrapolate functional implications of this activity. Here we perform quantitative binding experiments  SMAD family transcription factors are central mediators of the TGFβ superfamily signaling pathway ,2. The fIn addition to their canonical role in transcriptional regulation, several SMAD proteins have also been implicated in the processing of primary microRNA (pri-miRNA) transcripts via the recruitment of the microprocessor complex . AdditioThe parallels between SMAD RNA and DNA binding are intriguing, but the sequence and structural requirements for RNA binding are poorly defined. The crystal structure of SMAD3 bound to dsDNA revealed a mechanism of sequence recognition typical of transcription factors in which the protein reads a specific pattern of hydrogen bonds in the major groove of the B-form helix . The proHere, we describe the thermodynamic characterization of SMAD binding to RNA ligands. All work was performed using the SMAD3 protein, which is one of four SMAD proteins previously reported to bind RNA . ContrarEscherichia coli (Integrated DNA Technologies). SMAD3 MH1 (amino acids 1–132) and full-length SMAD3 (1–425) were PCR amplified and cloned into the pET SUMO vector according to manufacturer's recommendations (Thermo Fisher Scientific).SMAD3 was expressed and purified as an N-terminally tagged 6xHis-SUMO fusion protein prior to cleavage of the tag for biochemical characterization. A synthetic gene for full-length human SMAD3 was codon optimized for expression in E. coli. Cells were transformed with the pET His-SUMO-SMAD3 plasmid and grown at 37°C in luria broth supplemented with 50 μg/ml kanamycin to an OD600 of 0.5–0.6. Cells were then cold shocked on ice for 30 minutes with occasional shaking. Protein expression was induced by the addition of 0.5 mM isopropyl β-D thiogalactopyranoside (IPTG) and cells were incubated with shaking for 20 hours at 18°C. Cells were harvested by centrifugation at 5000 RCF for 10 min and resuspended in lysis buffer , and 5 U/ml benzonase (EMD millipore)).The His-SUMO-SMAD3 fusion protein was expressed in BL21 (DE3) Cells were lysed by passing through a microfluidizer three times at 15 000 PSI and insoluble material was pelleted by centrifugation at 15 000 RCF for 30 min. Soluble material was mixed with Ni-NTA agarose (Qiagen) preequilibrated in lysis buffer. The slurry was incubated for 1 h with gentle rocking at 4°C and poured into a flex column for purification by gravity flow. The column was washed twice with lysis buffer and once with wash buffer  before eluting with elution buffer . 6xHis-Ulp1 protease was added to the eluent and dialyzed overnight at 4°C in 6–8 kDa MWCO tubing (Spectrum Labs) in dialysis buffer .Following cleavage of the His-SUMO tag another Ni-NTA column was used to separate the cleaved His-SUMO tag and His-Ulp1 from SMAD3. The overnight dialysis product was poured over a preequilibrated Ni-NTA column, but SMAD3 was not present in the flow through. SMAD3 was eluted with 20 mM potassium phosphate pH 8.0, 300 mM sodium chloride, 20 mM imidazole pH 8.0 and 3 mM βME. Eluent was concentrated and purified further by gel filtration using a HiLoad 16/600 Superdex 200 column  equilibrated in 25 mM Tris–HCl pH 8.0, 115 mM potassium chloride, and 10 mM DTT. Fractions corresponding to highly pure, monomeric SMAD3 MH1 or full-length SMAD3 were pooled, concentrated to ∼100–250 μM, snap frozen in liquid nitrogen and stored at –80°C.12 were synthesized on a MerMade synthesizer (BioAotomation) using standard phosphoramidite chemistry and deprotected as previously described . Short RNA oligonucleotides under 20-nt and construct escribed .in vitro from synthetic ssDNA templates (Thermo Fisher Scientific). Equimolar amounts of ssDNA template and a universal T7 promoter sequence were annealed. One microgram annealed template was then used in a 100 μl transcription reaction containing 5 mM each rNTP, 22 mM magnesium chloride, 40 mM Tris–HCl pH 8.0, 2 mM spermidine, 10 mM DTT, 0.01% Triton X-100, 40 units RNase inhibitor (New England Biolabs) and 5 μl T7 RNA polymerase. Transcription reactions were incubated at 37°C for 2 h.Long RNA oligonucleotides were transcribed All RNA was gel purified by denaturing gel electrophoresis. Appropriate bands were excised and RNA was extracted by crush soak in 10 mM MOPS pH 6.0, 300 mM sodium chloride, and 1 mM disodium-EDTA. After extraction, RNA was ethanol precipitated, washed once with 70% ethanol and resuspended in water.32P] adenosine triphosphate (ATP) and T4 polynucleotide kinase (New England Biolabs) according to manufacturer's recommendations. Free ATP was removed using G25 spin columns  and oligonucleotides were purified by denaturing gel electrophoresis. Following purification and ethanol precipitation, RNA was resuspended in 25 mM Tris–HCl pH 7.5 and 115 mM potassium chloride and stored at –80°C.Oligonucleotides were 5′ end labeled for EMSA experiments. Transcribed RNAs were prepared for end-labeling by dephosphorylation with Antarctic Phosphatase (New England Biolabs) according to manufacturer's recommendations. Following dephosphorylation, enzyme was inactivated by heating to 70°C for 5 min. Oligonucleotides were 5′ end labeled using [ϒ-Double-stranded oligonucleotides were created by 5′ end labeling one strand and annealing it with 1.2 molar equivalents of the unlabeled complementary strand. Annealing reactions were performed with at least 170 nM each oligonucleotide in annealing buffer . Strands were annealed by heating to 95°C for 5 min and cooled by placing at room temperature.RNA was folded at low concentrations immediately prior to binding experiments to ensure the absence of dimers or other misfolded conformers. RNA was diluted to less than 10 nM in 25 mM Tris–HCl, 115 mM potassium chloride and 1 mM DTT. The RNA was then placed in a 95°C heating block and allowed to slow cool to room temperature over the course of 60 min.KDapp reflects the apparent macromolecular dissociation constant of the highest affinity interaction between SMAD3 and the oligonucleotide.For binding reactions, SMAD3 was serially diluted and mixed with trace labeled oligonucleotide at a concentration less than 5 nM. Binding buffer contained 25 mM Tris–HCl, 115 mM potassium chloride, 1 mM magnesium chloride, 1 mM DTT, 20 ng/μl low molecular weight poly I:C (Invivogen), 0.1 mg/ml bovine serum albumin and 10% glycerol. Reactions were incubated on ice for two hours (longer incubation had no affect on results) and loaded onto a 0.75 mm thick native gel made from 6% to 10% acrylamide and 0.5× TBE . Gels were run in 0.5× TBE at 100–120 V for 1 h at 4°C. Gels were dried on whatman paper, exposed to a phosphorimager screen overnight and scanned on a Typhoon FLA 9500 biomolecular imager . Data were quantified manually in ImageQuant  and corrected for background. Fraction bound was calculated as the total counts from all shifted species divided by the total counts from all bands in a lane. Thus, KDapp. Oligonucleotides that bound weakly, such as 12 and 16, were measured in duplicate and the initial pri-miRNA binding   . ProteinThe competition experiment was performed in a similar fashion to the EMSA binding experiments. 1 nM labeled dsDNA containing an SBE was mixed with 350 nM SMAD3 MH1 in binding buffer and incubated on ice for 1 h. Serial dilutions of unlabeled competitor RNA were made and mixed with the preincubated SMAD/dsDNA complex. Competition reactions were incubated on ice for 4 h to reach equilibrium before performing gel electrophoresis, as described for EMSA binding experiments.KDapp. Individual replicates were fit to Equation  were selected as design templates based on their reported interaction with SMAD and the presence of a canonical SBE  contained the core SBE and five flanking base-pairs from the predicted pri-miRNA secondary structure, so as to maintain local structural elements that might promote binding  and we mutated the SBE sequences of two designed RNAs (1b and 6b) . Th. Th1–8) g Figure . Five GC) Figure .19) . The DNA-binding domain MH1) of SMAD3 was used in our binding assays because of its reported ability to confer specificity for the SBE in RNA . We veri of SMAD3) Figure .3b and 4b) as well as a complete pri-miRNA stem loop sequence (3c) , and again, we saw no difference in affinity between RNAs with and without the SBE  Figure . We inclE Figure . ReplicaE Figure . Thus, SKDapp) , based on hsa-pri-miR-21, which contains the stem loop as well as the predominantly single-stranded flanking regions (11) from adenovirus (VAI) that is largely base-paired, but contains additional structural features that distinguish it from the RNA constructs tested to this point (10) binds with slightly higher affinity than the shorter constructs, demonstrating a length-dependent RNA binding activity (11) binds 6-fold more tightly than 10. Thus, SMAD3 can discriminate between length-matched RNAs and does not preferentially bind to pri-miRNAs.Our binding experiments revealed no sequence specificity for SMAD3 binding, but we did see slightly higher affinity for longer RNAs that approached a 1 μM apparent dissociation constant  Figure . Indeed,) Figure . We furt regions . We addiis point . As expeactivity . Surpris12), a set of double-stranded RNA and RNA/DNA hybrid duplexes (13 and 14), and a 30-basepair stem loop (16)  derives from domain four of a group IIC intron and comprises a stem loop with large internal loops  derives from domain three of a group IIB intron and comprises a four-way junction with a bulged stem   also binds with low affinity  increase affinity slightly, but the KDapp remains in the micromolar range and no clear shifted species is formed in the EMSA, suggesting the lack of a discrete, stable protein/RNA complex  Figure ,19. The s Figure . The secm Figure . SMAD3 b) Figure . Consist17 and 18) bind to SMAD3 with significantly higher affinity and they form discrete shifted species that are visible by EMSA  binds with a 190 nM KDapp while the perfectly base-paired stem loop (16) binds 20-fold weaker, despite being only nine nucleotides shorter  only two nucleotides different in length , in agreement with previously reported values . To determine if these binding activities are mutually exclusive, we performed competition assays in which unlabeled RNA or DNA was added to a pre-bound SMAD3/dsDNA complex  efficiently competes with canonical dsDNA binding at nanomolar concentrations consistent with the KDapp, while low-affinity RNAs (16) compete much less effectively. Thus, high-affinity RNA binding to SMAD3 competes with canonical dsDNA binding, suggesting that RNA and DNA bind to the same site on SMAD3.The apparent affinity between SMAD3 and these complex RNA structures is comparable to its canonical dsDNA binding affinity. We measured a s Figure  11). In. InKDapp19) forms a single shifted species with SMAD3  . Each of these constructs binds with sub-micromolar apparent affinity, but do not reach the affinity of 17. Additionally, there are fewer shifted species than with 17, consistent with a decrease in the number of SMAD3 binding sites  Figure . This trs Figure . Togethe20) and the Rev-response element from HIV (21), both of which contain internal loops that result in helices with expanded major grooves, and which mimic the recognition potential of B-form dsDNA. However, both of these RNAs bind weakly to SMAD3, with affinities that are even lower than 17c, which is similar length (Figure The specificity of SMAD3 for large internal loops and junctions raises the possibility that these RNAs mimic the canonical dsDNA ligand recognized by SMAD3. Large internal loops within RNA stems might be capable of forming distorted helices with expanded major grooves that mimic B-form dsDNA ,20. We th Figure . These rHere, we describe a novel, high-affinity RNA binding activity for the transcription factor SMAD3. SMAD3 binds RNAs with large internal loops or junctions with mid-nanomolar apparent affinity. This affinity for RNA matches that observed for a specific dsDNA ligand. Therefore, RNA binding has the potential to significantly impact SMAD3 function as well as that of homologous SMAD proteins.The majority of the human genome is transcribed into RNA, providing a wealth of potential SMAD binding partners . RelativThe current understanding of when and where SMAD3 exists in the cell suggests multiple ways in which RNA could interact with SMAD3 to regulate TGFβ signaling. Prior to signaling, SMAD3 is localized to the TGFβ receptor at the plasma membrane via direct interaction with the scaffolding protein SARA . TGFβ sivice versa, and consequently they may impact the genes that are regulated by TGFβ signaling. Previous experimental results have been interpreted under the assumption that SMAD is a DNA binding protein. However, our results suggest that RNA binding must also be considered. For example, ChIP experiments show SMAD enrichment at enhancer regions, despite the fact that the SBE is not the most enriched sequence motif (In the nucleus, SMAD localizes to the chromatin where a large number of nascent and mature RNAs are concentrated . These Rce motif ,34. Thisce motif ,36. ThusThe relatively high affinity and low specificity of SMAD3 for RNA suggests the need for regulation of this binding activity. Many of the most abundant cellular RNAs, such as rRNA, tRNA, 7sk, 7sl, snoRNA, snRNAs, and some mRNAs are present at intracellular concentrations near or above the apparent dissociation constant for SMAD3 –39. TherOur results have additional implications regarding the requirements for SMAD DNA binding. We have shown that multiple RNA binding sites achieve the same effective dissociation constant and compete with specific DNA binding. When localized to the chromatin, SMAD will encounter a large number of RNA binding sites . Therefoin vitro as indirect effects complicate cell-based experiments. The handful of transcription factors that have been characterized agree well with our observations on SMAD3. TFIIIA, NF-κB, RUNX1, GR, and now SMAD3 each bind RNA with a mid- to low-nanomolar apparent KD within an order of magnitude of their KD for DNA (The extrapolation of our findings to other transcription factors is still uncertain, but our results agree with the few transcription factors that have been well characterized. Several transcription factors have been suggested to bind RNA, but very few have been quantitatively characterized . Our res for DNA –47. TFII for DNA ,20,48,49The RNA binding ability of SMAD must be considered in the design and interpretation of future experiments. We have demonstrated a high-affinity interaction with RNA that is not sequence specific, as previously described, but instead has moderate specificity for internal loops or junctions. The magnitude of this interaction suggests a novel role for RNA in TGFβ signaling.Supplementary DataClick here for additional data file.


Until fairly recently, genome-wide evolutionary dynamics and within-host diversity were more commonly examined in the context of small viruses than in the context of large double-stranded DNA viruses such as herpesviruses. The high mutation rates and more compact genomes of RNA viruses have inspired the investigation of population dynamics for these species, and recent data now suggest that herpesviruses might also be considered candidates for population modeling. High-throughput sequencing (HTS) and bioinformatics have expanded our understanding of herpesviruses through genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures. Here we discuss recent data on the mechanisms that generate herpesvirus genomic diversity and underlie the evolution of these virus families. We focus on human herpesviruses, with key insights drawn from veterinary herpesviruses and other large DNA virus families. We consider the impacts of cell culture on herpesvirus genomes and how to accurately describe the viral populations under study. The need for a strong foundation of high-quality genomes is also discussed, since it underlies all secondary genomic analyses such as RNA sequencing (RNA-Seq), chromatin immunoprecipitation, and ribosome profiling. Areas where we foresee future progress, such as the linking of viral genetic differences to phenotypic or clinical outcomes, are highlighted as well. Herpesviruses infect and affect every human on the planet, with a universally penetrant global public health impact –4. Most –Here we review recent studies that have used high-throughput sequencing (HTS) and genome-wide analyses to explore the diversity and evolution of herpesviruses. In the first half of this review, we examine data suggesting that the diversity and evolution of herpesviruses are impacted by mechanisms extending beyond the usual consideration of polymerase fidelity. These include the influence of minority alleles and standing variation in the virus population, recombination between viral genomes, horizontal gene transfer, and nontemplated mechanisms such as ribosome frameshifting and RNA editing  and 2. W−7 or ∼1 × 10−8 mutations per base per infectious cycle , which relies on two viral antagonists to combat the host antiviral protein kinase R (PKR)  . After e R (PKR) . VariatiHorizontal gene transfer (HGT), or the movement of genetic material between unrelated organisms, provides another avenue for evolutionary adaptation. In the case of herpesviruses, at least 20% of the core genes shared by all herpesvirus subfamilies are surmised to have cellular origins, while others appear to have originated in another viral species –50. The –53–53–in vivo.Mechanisms of genetic variation such as single-nucleotide changes, recombination, and horizontal gene transfer are well accepted for their roles in the evolution of herpesviruses. Limited but exciting data suggest that other mechanisms, including several that are more often associated with RNA viruses, may also contribute to the diversity of herpesvirus coding potential. These include ribosome slippage, RNA editing, and novel transcripts revealed by RNA sequencing (RNA-Seq) and ribosome profiling or footprinting. These mechanisms may not be revealed by examining populations of viral genomes but may nonetheless influence phenotypes observed Ribosome frameshifting and RNA editing are two mechanisms by which herpesviruses can achieve a phenotypic outcome different from the outcome that would be predicted by analysis of the nucleotides encoded in their genome. These outcomes can be detected by examining viral transcripts or proteins but may otherwise go undetected in the comparison of genome sequences. Ribosome frameshifting is a regular feature of translation for retroviruses such as HIV, where it enables the production of nucleocapsid and polymerase from the same RNA transcript. Although it is less frequent, ribosome frameshifting has been demonstrated to occur on transcripts of thymidine kinase (TK) in HSV-1 , 60. MicFinally, HTS approaches have highlighted the presence of previously unrecognized coding potential in herpesvirus genomes, through the use of RNA-Seq and ribosome profiling approaches that demonstrate the shift from host to viral transcriptional and translational control during infection –68. Thes–64–Early applications of HTS to herpesvirus genomes focused on just one or two examples of a given species , using v30–70–73–As the genomic comparison of large DNA viruses from cultured stocks became more tractable, the goal of achieving similar resolution from uncultured viruses became a priority. The development of oligonucleotide enrichment methods has facilitated this goal for herpesviruses , 77, 81.Many of the studies described above that catalogued herpesvirus diversity have focused on defining the consensus genome of each new sample. The consensus genome represents the most common allele or nucleotide at each position . In the As for RNA virus genomes, deep coverage of large DNA virus genomes has enabled the detection of minority variants, or heterogeneous alleles, and their potential expansion in different environments . It was 17–HTS methods have brought the issue of viral population diversity to the fore. Almost all experiments conducted with herpesviruses utilize a population of genome-containing virions . The sam90–Homo sapiens/Texas, USA/1963/KOS-KOS63, indicating both the year and location of its origin  versus high (laboratory strain) number of passages that a viral population has undergone in cell culture—though in practice these terms are interpreted differently by every research group , 24. Thes origin , 94. This origin , 94.de novo assembly. Alignment-based approaches utilize prior genome knowledge to achieve a faster outcome, but these are prone to miscalling of minority and structural variants  and CLC Bio (Qiagen)  , 100. Th(Qiagen) , 103.The rationale for a strong foundation of high-quality genomes has been well established by the human genome project and multiple microbial genome projects . A wide Here we have focused on the several areas of recent progress in understanding the genomic diversity and evolution of human herpesviruses. These advances have been driven by the rapid expansion and application of HTS, bioinformatics, and comparative genomics in virology. Together, these data have reshaped our sense of the stability of herpesvirus genomes. While these viruses possess high-fidelity polymerases, their ability to accrue standing variation, and to undergo recombination with neighboring genomes, creates many opportunities for selective pressures to induce rapid genetic shifts. Examples of this include the expansion of minority variants in niche locations in congenitally infected infants and the selection of drug-resistant variants during antiviral therapy , 20, 86.First, we foresee the improvement and extension of third-generation sequencing and genome editing technologies to herpesviruses. Early applications of MinION and SMRT long-read sequencing to herpesvirology have shown promise in revealing novel transcriptional networks , 85 and 108–Finally, we consider the linking of viral genetic variation to observable phenotypes to be one of the greatest challenges for virology. The advance of HTS and genomics has begun to enable the application of genome-wide association studies (GWAS) and quantitative trait locus (QTL) approaches to herpesviruses , 112. Br


Stony coral cover decreased and macroalgal cover increased significantly with levels of snorkeling, while trends varied among taxa for other organisms such as gorgonians, fire corals, and sea urchins. We conclude that benthic organisms appear to exhibit taxon-specific variation with levels of recreational snorkeling. To prevent further degradation, we recommend limitation of snorkeler visitation rates, coupled with visitor education and in-water guides to reduce reef-damaging behaviors by snorkelers in high-use areas. These types of management activities, integrated with reef monitoring and subsequent readjustment of management, have the potential to reverse the damage potentially inflicted on coral reefs by the expansion of reef-based recreational snorkeling.Chronic anthropogenic disturbances on coral reefs in the form of overfishing and pollution can shift benthic community composition away from stony corals and toward macroalgae. The use of reefs for recreational snorkeling and diving potentially can lead to similar ecological impacts if not well-managed, but impacts of snorkeling on benthic organisms are not well understood. We quantified variation in benthic community structure along a gradient of snorkeling frequency in an intensively-visited portion of the Mesoamerican Barrier Reef. We determined rates of snorkeling in 6 water sections and rates of beach visitation in 4 adjacent land sections at Akumal Bay, Mexico. For each in-water section at 1–3 m depth, we also assessed the percent cover of benthic organisms including taxa of stony corals and macroalgae. Rates of recreational snorkeling varied from low in the southwestern to very high (>1000 snorkelers d Stony corals compete both among themselves and with other non-reef-building organisms for dominance on tropical coral reefs . NaturalEcotourism in the form of recreational diving and snorkeling has been hailed as a solution to the ecosystem degradation caused by extractive reef uses such as fishing and mining, because it incentivizes the preservation and non-destructive use of reef resources . The devWhen snorkelers and divers accidentally contact the reef with their bodies, fins, or other diving gear, they often cause damage by fracturing the skeletons and abrading the thin tissues of living stony corals ,24. ThisReduction in the abundance of herbivorous fishes also may lead to an increase in the abundance of sea urchins through competitive release, because both groups consume benthic algae . Sea urcThe Mesoamerican Barrier Reef System  is the Here we quantify variation in the benthic community structure of patch reefs in Akumal Bay with levels of in-water recreational snorkeling and on-land beach use by visitors. We analyze patterns of space use exhibited by stony corals, macroalgae, and other benthic organisms in relation to levels of snorkeling. We then discuss the implications of these patterns for the sustainable management of recreational snorkeling on coral reefs.http://ceakumal.org/). CEA issued the permission for access to the study locations. These field studies did not involve contact with any endangered or protected species.The present study was conducted during May to July 2015 on coral reefs in Akumal Bay, Quintana Roo, Mexico  to low in the southwest bay . Based 2 area), depth (1.3–1.7 m deep), and distance from shore (~ 120–200 m distant), to ensure that reefs exposed to roughly similar physical conditions were sampled within each section , depth (2.5–3.0 m deep), and distance from shore , we selected 2 patch reefs to quantify benthic community structure. Patch reefs were selected based on their similarity in size  were selected because they were sunny (rather than rainy which deterred people from visiting the bay), and also were dates during which the beach contained relatively low levels of washed-up macroalgae ssum sp. , which ossum sp. .-1, for the first 10–20 min of each 2-h period). One observer counted all visitors on each section of the beach, while the other simultaneously counted all visitors  in the adjacent inner bay water section, and in the 2 outer bay water sections. The coordinated counting of visitors on land and in the water prevented double counting of any who moved between sections during a given period. It was difficult to identify the presence of a snorkel on some in-water visitors, especially those located in the outer bay near the reef crest. However, most persons observed in the water appeared to use a mask and snorkel and thus to function as snorkelers (rather than as waders or swimmers), so here are referred to interchangeably as snorkelers or as in-water visitors.On each of the 5 selected dates, 2 observers (B. Renfro and a field assistant) walked the entire length of the beach area (from section D to A) at the start of every other daylight hour between sunrise and sunset  within 3 quadrats per transect x 10 transects per reef , or grouped as other stony coral species (identified using [Millepora), sponges, sea anemones, or zoanthids (zoantharians) [2 cell surveyed, see above. Non-living substrate was recorded in 3 categories: sand, rubble, or dead stony coral not colonized by turf algae. We examined the cells within each quadrat from directly overhead, so that horizontal percent cover was estimated for all categories, including for organisms that also extended vertically into the water column, such as some stony and soft corals and sea fans.We quantified benthic community composition on the 10 selected patch reefs by deploying 10 line transects parallel to shore on each reef. Each transect was 10–15 m long depending on patch reef shape, and was deployed parallel to the other transects ~1 m apart (methods adapted from ), beginnf after ,52,53). ,53. 2 qued using ). Macroaed using . Other sharians) ,56. No oDiadema antillarum, and other less-common sea urchins grouped together, after [Within each sampled quadrat, we also assessed the abundances of sea urchins . If morr, after . Reef crWe analyzed visitor abundances by focusing on the peak-use periods of 14:00 h on the beach and 16:00 h in the water see , to reveTo assess patterns of benthic community structure, we calculated the mean percent cover of each benthic category within each of the 10 transects examined per reef, by averaging values from the 3 quadrats examined per transect, to yield 10 benthic samples per reef. We ranked the 10 reefs from low to high in terms of their exposure to snorkelers in each water section of the bay, based on the snorkeler count data (see above), and the location of each reef within each section. The northern patch reef within each section received more snorkelers than did the southern reef, because it was closer to the adjacent section where higher relative snorkeler levels were observed. This ranking of reef sites allowed more fine-scale spatial analysis than did use of the absolute snorkeler count data, which were recorded only for each large section of the bay . BenthicDuring the morning hours on all observed days, visitor abundance was low both on land and in the water. Visitor abundance increased toward afternoon to a daily peak at 14:00 h on the beach and 16:00 h in the water, when 536 ± 74 and 414 ± 44 visitors were observed on land  and in the water , respectively, and then declined sharply toward sunset .During peak beach use at 14:00 h, visitor levels varied significantly among on-land sections of the bay . On-land visitor occupancy in the northeastern section D, where the public accessed the beach from the main road in Akumal, was significantly higher than in all 3 other beach sections . Overall, in-water snorkeler abundances appeared to vary strongly with time of day, distance from shore, and distance along the shore from the main public access point in the northeastern bay.Abundances of visitors in the water (snorkelers) were ~ 23% lower than those on the beach at all times of day, and varied significantly among in-water sections . In the most frequently-visited northeastern section J, 262 ± 33 snorkelers were observed in the water during peak use at 16:00 h, with > 1,000 snorkelers visiting this area over the course of an entire day . Snorkel2 values of the relationships increased , while macroalgal cover was ~ 2-20x higher (55.0–85.9%). Percent cover ranged 4.0–17.2% for other benthic organisms, and 6.0–20.8% for non-living substrates (N = 10 patch reefs). Live stony coral cover decreased significantly with snorkeler abundance; in contrast, macroalgal cover showed the inverse pattern of significant increase with snorkeler abundance on the reefs . Both trncreased .Diploria strigosa decreased significantly in percent cover with snorkeling level, while boulder star corals Orbicella annularis increased significantly  than did other types of stony corals, and did not vary significantly with snorkeler abundance. In contrast, stony corals with branching and plating (foliaceous) morphologies covered less reef area , and both decreased significantly with levels of snorkeling . These tficantly , howeverficantly . In contAcropora palmata and foliaceous thin leaf lettuce coral Agaricia tenuifolia decreased significantly with snorkeling level, although A. palmata did so only slightly, and only when considering variation among all reef sites. Colonies of branching finger coral Porites porites exhibited the opposite trend  decreased significantly with increasing snorkeling level ; erect calcified macroalgae  and other macroalgal types were rare. Turf algae and CMA both increased significantly in percent cover with levels of snorkeling, although CMA only slightly so. Crustose coralline algae (CCA) and erect fleshy macroalgae (FMA), while uncommon, both decreased significantly with snorkeling level . All butDiadema antillarum occurred at abundances of 0–3 individuals m-2 , and were most common on the offshore patch reefs (#1–2) as well as on those near the northeastern edge of the study site (reefs #8–10). Their abundance decreased slightly but significantly with levels of snorkeling (Arbacia punctulata and slatepencil urchins Eucidaris tribuloides) were observed; both of the latter species grouped together occurred at abundances of 0–2 individuals m-2. They exhibited the opposite trend to that of D. antillarum, in that their grouped abundance increased slightly but significantly with snorkeling level  thus may negatively impact many of the stony corals, potentially opening reef space for algal colonization and leading to these reefs becoming macroalgal-dominated, as known for some other coral reefs exposed to human pressure [This study reveals high rates of recreational snorkeling on some patch reefs at Akumal Bay, Mexico. Live stony coral cover decreased significantly and macroalgal cover increased significantly with increased levels of snorkeling visitation on reefs in this bay. The intensive snorkeling activity in some areas of the bay  to ~ 34pressure ) to 57 apressure ,60,61, wpressure , and stopressure . If the pressure , both thRecent completion of a beach resort hotel along the mid-bay likely contributed to expansion of the once clearly high- versus low-use sections , into a Orbicella annularis on some patch reefs was unexpected, given the decline of this species recently throughout the Caribbean [O. annularis alter their reproductive strategies to asexually reproduce more in areas of high disturbance [O. annularis recently have exhibited similar trends of increased but localized recruitment, supporting the idea that this species could be resilient and recovering in some regions of the Caribbean [Porites porites also occurred on patch reefs with high snorkeling levels, despite the tendency of their skeletons to fracture when kicked or bumped by visitors [P. porites dominate some disturbed coral reefs [Acropora palmata were once abundant at Akumal, as evidenced by their large branching skeletal remains on both the reef crest and deeper portions of the inner bay . Both A. palmata and staghorn corals A. cervicornis historically dominated reefs throughout the Caribbean Sea [Agaricia tenuifolia are weedy corals which colonize open space rapidly and may take over reefs after acroporids die off [A. tenuifolia can outpace rapid skeletal growth [Diploria strigosa have been predicted to dominate Caribbean reefs as “winners” in the face of growing anthropogenic disturbance [D. strigosa recruitment. Whatever the causes, our data on high variability among stony coral types in their relationships with snorkeling intensity on these reefs suggest that reef-builders may exhibit species-level complexity in their responses to human contact.The species-specific trends in stony coral cover observed here may have been caused by several factors. The relatively high percent cover of mounding coral aribbean –66. Howeturbance , which paribbean . Small bvisitors . Colonieal reefs , and areal reefs . Coloniebean Sea , but havbean Sea ,73, inclbean Sea ,49. The  die off –76. Howel growth ,77. Coloturbance ,78. Theiturbance , so the The patterns documented here in the cover of both turf algae and erect calcified macroalgae (CMA) are similar to those reported from other coral reefs exposed to intense human activity –82, and Diadema antillarum was highly variable within some of the examined reef sites, so it is not possible to make firm conclusions about impacts of snorkeler activity. These sea urchins were once major herbivores on reefs in the Caribbean, together with some fishes [D. antillarum urchins on the outer reef crest could be due in part to the presence of large A. palmata skeletons that provide more protective crevice habitat than available at inner bay sites, potentially exacerbated by snorkeler breakage of stony corals in the inner bay. In contrast, abundances of the 2 other sea urchin species exhibited patterns which could relate to their small body size and robust, short spines, making them relatively insensitive to disturbance by snorkelers. The latter urchins also likely require less complex habitat  for protection than do D. antillarum, allowing them to inhabit the relatively low-relief reefs of the inner bay. Studies are needed to further assess the potential effects of recreational snorkeling and related disturbances on sea urchins.The abundance of long-spined sea urchins e fishes ,87. Durie fishes ,88 inclue fishes . The relThe trends observed here for fire corals, gorgonians and zoanthids (zoantharians) indicate that these 3 types of reef cnidarians could be sensitive to reef disturbances caused by intensive snorkeling activity. Some studies have reported that gorgonians are not much affected by anthropogenic disturbances ,89, whilOther factors in addition to recreational snorkeling pressure potentially contributed to the trends in the benthic communities recorded here. The inner and outer bay sites differed slightly in depth below sea level (~ 1.5 m depth difference) and distance from shore , but even when outer bay sites were excluded from analyses, significant trends remained for many of the benthic organisms examined. Freshwater seeps in Akumal Bay also release nutrients from polluted groundwater in the adjacent watershed , leading-1, up to ~ 300,000 year-1) were 50 times higher than the carrying capacity estimated for coral reefs of ~ 5000–6000 guided dives site-1 year-1 [We conclude that the most intensively-visited coral reefs in Akumal Bay probably have exceeded their carrying capacity for recreational snorkeling, and that they exhibit low stony coral and high macroalgal cover relative to other parts of the bay. Levels of visitation in intensively-used section J  visitation rates within examined sections of Akumal Bay, Mexico, (b) percent cover of benthic organisms, and (c) abundance of sea urchins on coral reefs surveyed in the bay. See map (XLSX)Click here for additional data file.


RNA-sequencing (RNA-seq) has become the standard method for unbiased analysis of gene expression but also provides access to more complex transcriptome features, including alternative RNA splicing, RNA editing, and even detection of fusion transcripts formed through chromosomal translocations. However, differences in library methods can adversely affect the ability to recover these different types of transcriptome data. For example, some methods have bias for one end of transcripts or rely on low-efficiency steps that limit the complexity of the resulting library, making detection of rare transcripts less likely. We tested several commonly used methods of RNA-seq library preparation and found vast differences in the detection of advanced transcriptome features, such as alternatively spliced isoforms and RNA editing sites. By comparing several different protocols available for the Ion Proton sequencer and by utilizing detailed bioinformatics analysis tools, we were able to develop an optimized random primer based RNA-seq technique that is reliable at uncovering rare transcript isoforms and RNA editing features, as well as fusion reads from oncogenic chromosome rearrangements. The combination of optimized libraries and rapid Ion Proton sequencing provides a powerful platform for the transcriptome analysis of research and clinical samples. High-throughput next-generation sequencing has become a standard method to study the complexities of the genome and transcriptome. In particular, RNA-sequencing has the ability to provide multiple types of information, including gene expression levels, alternative RNA splicing, sequence variants such as inherited single nucleotide polymorphisms (SNPs), and post-transcriptional variants introduced by RNA editing as well as gene fusions resulting from chromosomal translocations. For example, recent studies have demonstrated the importance of changes in RNA editing  and alteGenome-wide assays have estimated that as many as 94% of human genes produce multiple RNA isoforms through alternative RNA splicing . HoweverTo be effective, measurements of alternative RNA splicing require high sensitivity to detect rare isoforms and even coverage across the entire lengths of transcripts so that all exons are measured accurately. Specialized library preparation methods have been developed to detect rare transcripts or to analyze individual human cells . HoweverJurkat T cells , a human T lymphoblastoid cell line derived from an acute T cell leukemia, were cultured in RPMI-1640 Medium  supplemented with 10% fetal bovine serum , Trypsin-EDTA, RPMI, phosphate buffered saline, NewBorn calf serum and antibiotics (A/A) . Cultures were maintained at 37°C, and split 2–3 times per week.Cytokine-mobilized CD34+ cells  were cultured in StemSpan™ SFEM II media  and supplemented with StemSpan™ CC110 expansion supplement  for CD34+ cells. Jurkat and CD34+ cells were collected 24 hours after plating.Cell counting was conducted with a glass hemocytometer and coverslip. Trypan Blue-treated cell suspension was used to apply to the hemocytometer to exclude live cells from staining. Total RNA was isolated from cells using RNeasy® Mini Kit  according to manufacturer’s instructions. An on-column DNase digestion step for further residual DNA removal was included. Extracted total RNA was transferred to −80°C and stored there until sample processing.The Ribogone kit  was used to deplete ribosomal and mitochondrial RNAs. The RiboGone exclusively targets the depletion of 5S, 5.8S, 18S, and 28S nuclear rRNA sequences as well as 12S mtRNA sequences on the basis of hybridization and RNase H digestion. The Clontech SMARTer Universal Low Input RNA kit was used to prepare cDNA and the library was prepared using the Ion Plus Fragment Library kit .5’-GTGAGTGATGGTTGAGGTAGCAGTTCAGNNNNNN-3’. cDNA was then linearly amplified using suitable primers for linear amplification as described in the original MALBAC method (16); MALBAC primers also contained the Ion A adaptor sequence and an IonXpress barcode. Final amplification of the library used primers that ligate to the Ion A adaptor and key sequence and to a truncated Ion P1 adaptor. The final library was size-selected using E-Gel SizeSelect Agarose Gels .Ribosomal RNA was depleted with the Low Input RiboMinus Eukaryote System v2 kit . RiboMinus Magnetic Bead Cleanup  was used to concentrate and purify the rRNA-depleted RNA. In the MALBAC method, cDNA was made using primers containing random hexamers and an adaptor sequence: Total RNA was ribo-depleted with Low Input RiboMinus Eukaryote System v2 . RiboMinus Magnetic Bead Cleanup  was used to concentrate and purify the rRNA-depleted RNA. Ion Total RNA-Seq Kit v2 was used to make cDNA, add barcodes, and amplify the library. All cleanup steps were performed using Agencourt Ampure XP beads .RNA libraries were sequenced on P1v2 chips using the Ion Proton™ System . Sequencing was completed by the Analytical and Translational Genomics Shared Resource at the University of New Mexico Cancer Center. All samples were aligned with Torrent Mapping Alignment Program (TMAP), using the 1000 genomes GRCh37 phase II d5 the human reference genome. Subsequently, the data were aligned with the STAR aligner  using thCufflinks  with theThe total number of sequenced reads varied by technique and sample. Therefore, to avoid bias towards samples with more total reads, each sample was analyzed as the average of 5 randomly selected subsamples of 10 million reads. The subsampling was done without replacement using a reservoir sample tool . Each tiThe STAR aligned BAM files for total reads or the 10 million read subsamples were used to calculate the gene and isoform expression count using cufflinks version 2.2.1 . The couhttps://github.com/scottness/rna_seq_libraries_analysis). We excluded any splice junction sites from the data analysis that did not contain a read coverage of at least 5 and were not identified in all 5 subsamples. The splice junction sites were then compared across each method to identify the unique and shared junctions that are depicted in Venn diagrams.To compare splice sites that were shared or unique to samples, we combined the chromosome, first-intron base, and last-intron base information from STAR alignment files to make a unique identifier that could be compared across all samples. This script is available as ‘spliceJunctionTable’ . To filter out potential false positives in our data sets, we excluded any sites from our analysis that did not contain at least a minimum of 5 reads in all of the sampling groups for a given method. To exclude known SNP and mutation sites from our data, we filtered against the NCBI dbSNP Build 137 [The RNA editing sites were identified in the study using a modified variant caller format program, ‘editing_site_coverage_example’  used a random 8 nt binding sequence and 27 nt self-complementary common sequence designed to form loops after cDNA synthesis to encourage linear amplification and prevent subsequent exponential amplification that could skew the results. This approach has been described for amplifying and sequencing rare sequences or performing single-cell RNA sequencing . The DirWe first measured the extent of coverage across transcripts by identifying the 10,000 most highly expressed genes, summing the average coverage, and plotting the results . Both thTo gain a deeper understanding of the differences between the library methods, we analyzed features of the RP and DL results in detail. We compared the results from 6 different sequencing runs on the Ion Proton platform: CD34+ cell RNA with libraries prepared with either the RP or DL methods, all of which yielded 10–20 million mapped reads per library, or Jurkat T-cell line RNA prepared similarly, which yielded 20–30 million mapped reads per library . The excWe first analyzed the fraction of reads from each library marked as secondary alignments, indicating that they aligned with more than one position in the genome . AlthougWe also plotted the fraction of duplicate reads, defined as having identical start positions and CIGAR strings . The RP These two quality control metrics, secondary reads and duplicate reads, showed important differences between the ribosomal RNA depletion kits and the amplification approaches that have little impact on gene expression measurements, but may have important implications for assessing more advanced transcriptome features, such as alternative RNA splicing.To test the usefulness of the libraries for transcriptome analyses, we analyzed the total number of genes that were detected (as expressed above a base threshold) and the total number of different splicing variant isoforms detected in each library. Using the total data set from each sequencing run showed that both the RP and DL methods detected approximately 15,000 expressed genes in the CD34+ sample. As shown in , both ofWe observed very similar results when counting the number of different splice variant isoforms detected in each library. Both the RP and DL methods detected over 40,000 transcript isoforms in the CD34+ samples. The RP method detected over 60,000 isoforms in the Jurkat samples. The first DL library, DL-3, detected only 27,000 isoforms in Jurkat, but using 4 times more reads (DL-4) led to the detection of nearly 80,000 transcript isoforms in Jurkat cells. Thus, increasing the number of reads increases the sensitivity of the transcriptome assays and the number of expressed genes or isoforms detected. However, using more reads also increases the cost of the RNA-seq assays.As shown in To check for sampling errors, we randomly sampled each library 5 times, generating independent 10 million read subsamples, then averaged the gene and isoform detection results. The results, shown in , show thThe results of these assays are summarized in , by showRNA editing results from post-transcriptional enzymatic modifications lead to apparent changes in the RNA sequence. Changes in RNA editing have recently been implicated as important for cell differentiation and in cancer , 29. We We also tested whether the methods detected the same or different sets of RNA editing sites. As shown in the Venn Diagrams in The human MYB gene can produce as many as 60 different RNA isoforms through alternative RNA splicing, which encode at least 20 different versions of the Myb protein , 8. We cFinally, we analyzed the numbers and locations of MYB transcript isoforms detected by each RNA-seq library method. We started with 10 million reads selected from each data set and filtered the RNA-seq results to limit our analysis only to junction reads, identified as spanning two or more exons, and plotted the results in the “Sashimi Plots” shown in We tested several RNA-seq library preparation methods, using both popular commercial kits and homemade approaches, to find the advantages and disadvantages of each and to determine how best to analyze advanced features of the transcriptome, such as RNA editing and alternative RNA splicing. One of the most important findings was a strong correlation between the total number of reads analyzed and the sensitivity of the assays, measured in terms of expressed genes or isoforms detected. This relationship is well known and is reflected in the ENCODE Consortium best practices guidelines , which aWe found a major difference in the results obtained when using different ribosomal RNA depletion kits. The kit recommended for use with the DL approach did not remove the very abundant snoRNAs, and those sequences consumed a relatively large fraction of the resulting RNA-seq libraries. For researchers focused primarily on protein-coding genes, the use of a different ribosomal RNA depletion method, such as the kit we used in the RP method, would be a better approach. However, this demonstrates how choices that seem relatively innocuous, such as the choice of ribosomal RNA depletion kit, can have a major impact. On the other hand, data sets generated using kits that remove snoRNAs cannot be used for some purposes where analysis of snoRNAs is important.Another difference in the methods is whether they preserve the strand information of the transcripts. For many purposes, such as gene expression measurements, preservation of strand information may not be important, but it is critical for measuring transcripts from overlapping genes. The RP method we used was better at most metrics that we analyzed but did not conserve the strand information, which is a drawback. The DL method did preserve the strand information for transcript reads but had other disadvantages. These issues should be considered carefully by investigators before designing an RNA-seq approach.Our analysis of MYB gene alternative splicing detected at least 29 different transcript isoforms in both CD34+ hematopoietic progenitors and in Jurkat T-cells, which is consistent with previous reports , 8. Howe