To further investigate sources of bias in miRNA sequencing, we compared the over- and under-represented reference set miRNAs in datasets obtained by TGIRT-seq NTT and 4N ligation protocols (Fig. (A) RNA-seq saturation curves. 3E). To address this difficulty, we designed a new R2R adapter (denoted NTT) in which a single T residue was inserted at position 3, thereby replacing the favored C at this position with a disfavored T, but leaving the remainder of the R2R sequence unchanged (Fig. The bias corrector uses the first and last 3 nucleotides of each miRNA to predict the measurement errors, so that a corrected abundance can be computed by subtracting the predicted measurement error from the experimentally determined abundance of each miRNA (see Methods). and D.C.W. Transcription. For the 3 bias, we first thought that the preference for a G residue and against a U residue at position 1 might reflect the strength of the base-pairing interaction between that nucleotide and the 3-overhang nucleotide of the DNA primer that is used to direct TGIRT template-switching, with a strong rG/dC base pair favored over a weak rU/dA pair. The 5-end bias in TGIRT-seq is due in large part to sequence biases of the thermostable 5 App DNA/RNA ligase used for single-stranded ligation of the R1R adapter to the 3 end of the cDNA (Fig. Ramskld, D. et al. Use a strand of DNA to build a molecule of mRNA. 11, a032375, https://doi.org/10.1101/cshperspect.a032375 (2019). For datasets obtained for the Miltenyi miRXplore miRNA reference set, reads obtained using the NTT and NTC adapters were processed as described above for UHRR datasets and then mapped with Bowtie2 using local alignment with default settings to the Miltenyi miRXplore reference sequences. Genome Biol. Likewise, the normalized abundances (transcripts-per-million; TPM) of ERCC spike-ins from the TGIRT-seq datasets correlated well with the expected spike-ins inputs (=0.98; Supplementary Fig. UV DNA damage results in bulky DNA adducts - these adducts are mostly thymine dimers and 6,4-photoproducts. 43, e2, https://doi.org/10.1093/nar/gku1235 (2015). 7, 709715 (2010). J Mol Biol. Nat Methods. volume9, Articlenumber:7953 (2019) For the 5-ligation bias, we noted that established small RNA-seq methods that employ T4 RNA ligases I and II to sequentially ligate adapters to the 5 and 3 ends of RNAs or cDNAs benefit from employing DNA adapters with four randomized nucleotides at the ligated ends (referred to as 4N protocols), with such adapters giving lower bias and better coverage at low sequencing depths than those with invariant sequences at their ends33,34,35,36. GsI-IIC RT (TGIRT-III) has been used for a variety of applications, including the comprehensive profiling of whole-cell, exosomal and plasma RNAs7,8,13,14; quantitative tRNA-seq based on the ability of the TGIRT enzyme to give full-length end-to-end reads of tRNAs with or without demethylase treatment8,15,16; determination of tRNA aminoacylation levels17; high-throughput mapping of post-transcriptional modifications by distinctive patterns of misincorporation13,15,16,18,19,20; identification of protein-bound RNAs by RIP-Seq or CLIP18,21; and RNA-structure mapping by DMS-MaPseq22,23 or SHAPE24. 5B). In the TGIRT-based version of this method, template-switching rather than RNA ligation is used to add an adapter containing both R1R and R2R sequences, and the resulting cDNAs with the linked R1R/R2R adapter are gel-purified and circularized with CircLigase for RNA-seq library construction6,18. S8). BMC Genomics. The black boxes in the violins indicate the interval between first and third quartiles, and the vertical lines indicate the 95% confidence interval for each method. Curves were truncated at 3 million reads. A highly proliferative group IIC intron from Geobacillus stearothermophilus reveals new features of group II intron mobility and splicing. Nottingham, R. M. et al. Other library preparation methods (gray lines) include NEBNext, TruSeq and CleanTag. As noted previously by Giraldez et al.36, biological samples would likely behave differently from synthetic RNA pools tested at a single concentration in vitro. A study comparing TGIRT-seq to benchmark TruSeq v3 datasets of rRNA depleted (ribo-depleted) fragmented Universal Human Reference RNA (UHRR) with External RNA Control Consortium (ERCC) spike-ins showed that TGIRT-seq: (i) better recapitulates the relative abundance of mRNAs and ERCC spike-ins; (ii) is more strand-specific; (iii) gives more uniform 5- to 3-gene coverage and detects more splice junctions, particularly near the 5 ends of genes, even from fragmented RNAs; and (iv) eliminates sequence biases due to random hexamer priming, which are inherent in TruSeq7. Figure 3 & Wiener, M. Classification and regression by randomForest. 1C). Nature. Science. ViennaRNA package 2.0. Unmapped reads from Pass 1 were re-mapped to Ensembl GRCh38 Release 76 by Bowtie 250 v2.2.6 with local alignment to improve the mapping rate for reads containing post-transcriptionally added 5 or 3 nucleotides (e.g., CCA and poly(U)), short untrimmed adapter sequences, or non-templated nucleotides added to the 3 end of the cDNAs by the TGIRT enzyme (denoted Pass 2). The recently determined crystal structure of full-length GsI-IIC RT in an active conformation with bound substrates12 provides a platform for detailed analysis of the structural basis and possible alleviation of this 3-end bias. Shurtleff, M. J. et al. Article Uniquely mapped reads with lengths between 15 and 40 nt (8688% of the mapped reads for the NTT adapter; Supplementary TableS2) were retrieved and used to calculate the counts table for 962 miRNAs. 24, 183195 (2018). conducted bioinformatic analysis. Burke, J. M. et al. Once flanking the region to be sequenced, the adapters provide primer annealing sites, first for the reverse transcription (RT) primer and later for the . miRNA count tables for 4N ligation, NEXTflex, TruSeq, NEBNext and CleanTag were downloaded from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA accession number SRP12684536), and counts from the 962 Miltenyi miRXplore RNAs were extracted for the comparisons. Nat Methods. 1B). 4AC, left panels). 1A), a step that can result in the differential loss of sequences corresponding to miRNAs and other very small RNAs, whose library products are close in size to adapter dimers (146 and 124 nt, respectively)14. 22, 111128 (2016). The plots showed that the 5- and 3-end sequence biases are similar for the NTT and NTC adapters, with the 5-RNA end bias for G or U and against A at position +3 being the reciprocal of the sequence preferences of the thermostable 5 App DNA/RNA ligase for the 3 end of the cDNA (see above), and the 3-RNA end bias against U and for G at position 1 including a contribution from TGIRT template-switching. PubMed Central Next, we used TGIRT-seq of miRNA reference sets to analyze and correct 5- and 3-end biases in miRNA-seq. S7B), confirming the importance of the first three 5- and 3-end positions compared to the internal positions. Nucleus. 26, 841842 (2010). PubMed Central 1. A k-fold cross-validation test of the random forest regression model in which the 962 miRNAs were divided into 8 subgroups, each of which was tested with a model trained on the remaining subgroup, gave R2 values of 0.46 to 0.66 (Supplementary Fig. However, the largest contributor for under-represented miRNAs and second largest for over-represented miRNAs was the 3 terminal nucleotide (position 1), which favored a G residue and disfavored a U residue (Fig. Additionally, we developed biochemical and computational methods for remediating 5- and 3-end biases, the latter based on a random forest regression model that provides insight into the contribution of different factors to these biases. By fitting the data to a random forest regression model, we found that the position-specific nucleotide preferences at the first three nucleotides from the 5 and 3 ends of the miRNA account for 81% (R2=0.81) of the measurement errors (Fig. The NTT and NTC primer mixes contain an equimolar mix of R2R DNAs with 3 A, C, G, and T residues. (E) Aggregate nucleotide frequencies at the beginning of Read 1 (5-RNA end; positions 1 to 14) and Read 2 (3-RNA end; positions 1 to 14) in combined datasets for technical replicates obtained by TGIRT-seq of fragmented UHRR plus ERCC spike-ins with either the NTC or NTT adapter (datasets NTC-F1 to F3 and NTT-F1 to F3, respectively). To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Picelli, S. et al. ADS RNA polymerase 3. 5- and 3-end nucleotides are color coded as indicated in the Figure. Li, S. et al. The datasets generated and analyzed in the current study are available in the National Center for Biotechnology Information Sequence Read Archive under SRA accession number SRP168562. Cite this article. Genes Dev. This likely reflects that the biases corrected by the two methods are orthogonal. 5A). Nucleic Acids Res. Meanwhile, the chemical knockdown strategy with molecular glue may promote innovative transcription factor degrader development in cancer therapy. 8B). Mapping and quantifying mammalian transcriptomes by RNA-Seq. Unused R2R adapters that are carried over from previous steps are also ligated to the R1R adapter by the 5 App DNA/RNA ligase (New England Biolabs), resulting in the formation of adapter dimers (pathway at right), which are removed by AMPure beads clean-up prior to sequencing. The ligase commonly used is T4 DNA ligase, which was first isolated from E. coli that were infected with the lytic bacteriophage T4. The TGIRT-seq correction for 5-end bias addresses sequence preferences of the ligase, which are larger for the 5 App RNA/DNA ligase than for the T4 RNA ligases used in the 4N protocols 33,34 . Safra, M. et al. S4). & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. In the meantime, to ensure continued support, we are displaying the site without styles Two-dimensional kernel density estimation of the distribution for miRNA abundances and lengths (n=962) is shown. Ozsolak, F. & Milos, P. M. RNA sequencing: advances, challenges and opportunities. Nucleic Acids Res. Another example of manipulation of the interaction of an E3 ligase and a transcription factor involves the use of thalidomide and its derivatives in the treatment of multiple myeloma. Google Scholar. The ligated products were purified by using a MinElute Reaction Cleanup Kit and amplified by PCR with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific; denaturation at 98C for 5sec followed by 12 cycles of 98C 5sec, 60C 10sec, 72C 15sec and then held at 4C). The template-switching reactions were incubated for 15min at 60C and then terminated by adding 1 l 5M NaOH to degrade RNA and heating at 95C for 5min followed by neutralization with 1 l 5M HCl and two rounds of MinElute column clean-up (Qiagen) to decrease the amount of unused R2R DNA adapter. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Thank you for visiting nature.com. The stacked bar graphs show the percentages of miRNAs having A, C, G, and U 3-end nucleotides, color coded as indicated in the Figure, in the datasets obtained with different ratios of 3-overhang nucleotides. Proc Natl Acad Sci USA 111, 1202512030 (2014). 1. RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. H.X. The gap is filled by DNA ligase, an enzyme that makes a covalent bond between a 5'-phosphate and a 3'-hydroxyl group ( Figure 3 ). . The mechanism of action of thalidomide in this disease was unclear until it was shown to enhance the binding of the E3 ligase cereblon (CRBN) to the Ikaros . The site-directed mutagenesis based on the PCR with a pair of inverse primers is relatively complicated; it includes PCR, phosphorylation, ligation with T4 DNA ligase, and transformation 12,13 . (C) Bioanalyzer traces comparing adapter-dimer formation using the previous NTC and improved NTT R2R adapters. TGIRT-seq libraries were prepared from (A) 40-nt or (B) 20-nt RNA oligonucleotides using the workflow of Fig. The PCR products were cleaned up by using Agencourt AMPure XP beads (1.4X volume; Beckman Coulter) and sequenced on an Illumina NextSeq 500 instrument to obtain 275-nt paired-end reads. The datasets obtained using the NTT and NTC adapters showed no substantial differences in the profiles of reads mapping to different genomic features (Fig. Only the first 3 bases (i=1 to 3) and the last 3 bases (i=3 to 1) of each miRNA were considered in this model. Effect of 5- and 3-end sequences on the representation of miRNAs in TGIRT-seq datasets. 2). DNA ligase Types of Nucleic Acids Nucleic. 4AC) or of the aggregate nucleotide frequency as a function of position from the beginning of Reads 1 and 2 (Supplementary Fig. Principal component analysis (PCA) based on the first 3 nucleotides from the 5 and 3 ends of the miRNA showed that the over- and under-represented miRNAs were almost linearly separable along the first principal component (PC1) of the PCA biplot (Fig. Overall, the most active variants were p50-ligase (i.e. By contrast, because TGIRT-seq employs a thermostable ligase for a single-stranded ligation of a DNA adapter to a cDNA at high temperature, any bias resulting from base-pairing interactions between the adapter and acceptor cDNA may already be minimal. Transcription lesions encountered on one copy, even if leading to degradation of a few Pol I complexes, will bear negligible effect on the total RPA194 . PMID: 8608452 PMCID: PMC1369371 Abstract Large quantities of RNA for study by NMR and X-ray crystallography can be produced by transcription reactions in vitro using T7 bacteriophage RNA polymerase. By avoiding gel-purification steps, the TGIRT Total RNA-seq method enables the rapid construction of comprehensive RNA-seq libraries containing nearly all RNA biotypes from small amounts of starting materials with less overall bias than other transcriptome-profiling methods7,8,14. Reads were then mapped by using HISAT249 v2.0.2 with default settings to a human genome reference sequence (Ensembl GRCh38 Release 76) combined with additional contigs for 5S and 45S rRNA genes and the E. coli genome sequence (Genebank: NC_000913) (denoted Pass 1). Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. Base-resolution mapping reveals distinct m1A methylome in nuclear- and mitochondrial-encoded transcripts. Alan M. Lambowitz. To identify other factors that might have contributed to the biased representation of these outlier miRNAs in TGIRT-seq, we defined over- and under-represented miRNAs as those whose log10CPM values after computational correction for end biases were 2 standard deviations higher (n=8) or lower (n=27) than the mean log10CPM, and then compared several potentially bias-inducing characteristics of these miRNAs to the remaining 927 more uniformly represented miRNAs (those in the center box in Fig. Library construction for next-generation sequencing: overviews and challenges. The proportion of uniquely mapped reads was higher for the NTT adapter than the NTC adapter (8688% and 6374%, respectively), possibly reflecting that the multiple rounds of AMPure bead clean-up required for the NTC adapter resulted in differential loss of miRNA-sized products, whose sequences map uniquely to the miRNA reference sequences, compared to larger aberrant products resulting from multiple template switches, whose sequences map to multiple loci. The first two principal components are shown. PLoS One. Hengyi Xu, Jun Yao and Douglas C. Wu contributed equally. However, we also found that nucleotides at some 5- and 3-end positions of the miRNAs in the reference set are correlated, in some cases with 2-test -log10 p-values>10 (e.g., 42% of the miRNAs with a disfavored A at position +3 have a disfavored U at position 1; Supplementary Fig. TGIRT-seq libraries were prepared from the Miltenyi miRXplore miRNA reference set containing 962 equimolar human miRNAs (Supplementary TableS2 and Methods). and JavaScript. 17, 1012 (2011). The TGIRT-seq method currently used for comprehensive transcriptome profiling (also referred to as TGIRT Total RNA-seq method) is outlined in Fig. Bioinformatics. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application. Article Here, we addressed two issues in TGIRT-seq library preparation, the disproportionate loss of miRNA sequences during AMPure beads clean-up of adapter dimers, and sampling biases resulting from 5- and 3-end sequences preferences in the ssDNA ligation and TGIRT template-switching steps. Hu, W. S. & Hughes, S. H. HIV-1 reverse transcription. TGIRT-seq libraries were prepared as described7,8 using 6ng of fragmented Universal Human Reference RNA (UHRR) with ERCC spike-ins or 50nM Miltenyi miRXplore RNA prepared as described above. Scatter plots comparing the representation of RNAs in technical replicates obtained using the NTT and NTC adapters gave Spearmans correlation coefficients () of 0.950.96 (Supplementary Fig. Thus, a preferable approach may be to use an alternative method for 5-adapter addition, such as leveraging the ability of TGIRT-III to add non-templated A residues to the 3 of cDNAs to enable template-switching to an acceptor oligonucleotide with 3 U residues, analogous to the Clontech/Takara SMART-seq protocols43,44. The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution. Google Scholar. For comparison, raw sequencing reads from published TGIRT-seq datasets generated from similarly prepared fragmented UHRR samples using the NTC adapter7 were downloaded (NCBI SRA accession number SRP066009) and processed using the same bioinformatic pipeline. Because some of the published datasets contain additional miRNAs, we created in silico subsamples containing only the 962 reference set miRNAs from each dataset for these comparisons. 5C,D). 24, 950965 (2018). Ligation reactions. PubMed For published datasets containing additional miRNAs, in silico subsamples containing only the 962 reference set miRNAs were used for the comparisons. The latter can base pair to the 3 end of the target RNA, serving as a springboard for TGIRT template-switching and the initiation of cDNA synthesis6. Collectively, our findings open up a new direction for transcription factors degradation by targeting atypical E3 ligase ZFP91. & Krug, R. M. Avian influenza virus PB1 gene in H3N2 viruses evolved in humans to reduce interferon inhibition by skewing codon usage toward interferon-altered tRNA pools. Log10CPM values for each miRNA in combined TGIRT-seq NTT datasets (n=3) are plotted against those in combined datasets for 4N protocols (n=24; Gilardez et al.36). the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Transcription and Translation. Cold Spring Harb Perspect Biol. NTTc and NTCc denote TGIRT-seq datasets obtained using the NTT or NTC adapters that were computationally corrected using the random forest regression model trained with the combined NTT datasets (Fig. 10, 10961098 (2013). Smart-seq2 for sensitive full-length transcriptome profiling in single cells. 30, 777782 (2012). By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (12ng input RNA). Qin, Y. et al. The standard approach to sequencing small RNA is to prepare a cDNA library by sequentially ligating 3' and 5' adapter DNA oligonucleotides of known sequence to the ends of the RNA [ 1-3 ]. Despite the similarities in every aspect of the catalytic process, the RNA ligase differs from the DNA ligase in that the RNA ligase uses ssRNA molecules to align and join, whereas the DNA ligase requires a duplex structure. Science. We found that this bias could not be mitigated by using an R1R adapter with randomized nucleotides near its 5 end, as in 4N ligation RNA-seq protocols, but could be corrected computationally by using a random forest regression model to give the same level of bias as in 4N protocols. Shen, P. S. et al. https://github.com/wckdouglas/tgirt_smRNA, https://doi.org/10.1101/cshperspect.a006882, https://doi.org/10.1101/cshperspect.a032375, https://doi.org/10.1128/microbiolspec.MDNA3-0050-2014, https://doi.org/10.1016/j.molcel.2017.10.019, https://doi.org/10.1371/journal.pone.0126049, https://doi.org/10.1371/journal.pone.0167009, http://creativecommons.org/licenses/by/4.0/, Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing, Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2, A method for simultaneous detection of small and long RNA biotypes by ribodepleted RNA-Seq, Exploring the expanding universe of small RNAs, TRMT6/61A-dependent base methylation of tRNA-derived fragments regulates gene-silencing activity and the unfolded protein response in bladder cancer, Cancel Katibah, G. E. et al. (B) Taking into account known biases of the 5 App DNA/RNA ligase7,28,29, the R2R adapter used previously in TGIRT-seq (denoted NTC) was modified by inserting a single T-residue at position 3, creating a modified R2R adapter (denoted NTT), which decreases adapter-dimer formation. Ligase, an enzyme that uses ATP to form bonds, is used in recombinant DNA cloning to join restriction endonuclease fragments that have annealed. 3D). contributed to the design of the experiments, analysis of data, and writing of the manuscript. Reactions were set up with all components except dNTPs, pre-incubated for 30min at room temperature, a step that increases the efficiency of template-switching and reverse transcription, and then initiated by adding dNTPs (final concentrations 1mM each of dATP, dCTP, dGTP, and dTTP). First, to address the adapter dimer problem, we used the known sequence biases of the thermostable 5 App DNA/RNA ligase employed for R1R adapter ligation to design an R2R adapter with a single nucleotide change that strongly decreases adapter dimer formation during TGIRT-seq library preparation (8899% lower compared to the previous NTC adapter; Fig. 8). RNA. TGIRT-seq of the Miltenyi miRXplore miRNA reference set using the NTT or NTC adapters and comparison of different methods for mitigating 5- and 3-end biases. ISSN 2045-2322 (online). Evans, M. E., Clark, W. C., Zheng, G. & Pan, T. Determination of tRNA aminoacylation levels by high-throughput sequencing. Nucleotide excision repair (NER) is a particularly important excision mechanism that removes DNA damage induced by ultraviolet light (UV). In the transcription stage a strand of DNA . In the first step, TGIRT enzyme binds to an artificial template-primer substrate comprised of an RNA oligonucleotide containing an Illumina R2 sequence with a 3-end blocking group (3SpC3) annealed to a complementary DNA oligonucleotide (R2R) that leaves a single nucleotide 3 overhang, which can direct template-switching by base pairing to the 3 end of an RNA template. Counts from each dataset were median normalized, log2 transformed, and used to generate scatter plots, empirical cumulative distribution function (ECDF) plots, and nucleotide frequency plots in R. RMSE was calculated using log2 transformed median normalized counts. Levin, J. 9. The resulting cDNA with an R2R adapter attached to its 5 end is incubated with NaOH to degrade the RNA template and neutralized with HCl, followed by two rounds of MinElute clean-up using the same MinElute column (Qiagen). Nat Methods. Sci Rep 9, 7953 (2019). Unlike retroviral RTs, which have been studied extensively and optimized for biotechnological applications for decades, the recently introduced TGIRT enzymes and TGIRT-seq methods are potentially subject to substantial improvement. Quantitative miRNA expression analysis: comparing microarrays with next-generation sequencing. Here, the term will be used for DNA-ligases, which play a central role in DNA replication, recombination, and repair. 4AC). Google Scholar. Google Scholar. Additionally, both the 5- and 3-biases may include a contribution from the RNA fragmentation process31. 687, 113134 (2011). Methods Mol Biol. 8). For example, DNA ligase can join two complementary fragments of nucleic acid by forming phosphodiester bonds, and . Asterisks on the top of the violins indicate significance of the difference between the outliers and remaining miRNAs determined by Wilcoxon test (*p=0.03; **p=0.004). The authors acknowledge the Texas Advanced Computing Center (TACC) at the University of Texas at Austin for providing high performance computing resources that have contributed to the research results reported within this paper. The name of the dataset is indicated below. Reverse transcriptases generate complementary DNA (cDNA) from a transcript. To assess sampling biases of the miRNAs in the TGIRT-seq datasets, we combined the 3 technical replicates for each adapter and compared the representation of miRNAs in the combined datasets to that in the miRNA reference set (Fig. Saturation curves and differences in coverage for the 962 miRNAs in the Miltenyi miRXplore miRNA reference set for TGIRT-seq with or without different bias correction compared to published datasets for established small RNA-seq methods. (B) Violin plots of miRNA abundance in datasets obtained by different methods. S9). Question: Are the following enzymes involved in DNA replication, transcription, or both? Cold Spring Harb Perspect Med. Bioanalyzer traces of TGIRT-seq libraries constructed from varying amounts of different-sized RNA oligonucleotides using either the NTC or NTT adapter. Analysis of TGIRT-seq datasets obtained for fragmented UHRR or plasma DNA suggested that a major source of sequence bias is the DNA ligation step using the thermostable 5 App DNA/RNA ligase, which has a preference for A or C and against U/T at position 3 from the 3 end of the acceptor nucleic acid7,28,29. Whatever the cause, nearly all of the sequence biases in TGIRT-seq libraries prepared from the fragmented UHRR were confined to the first 3 positions from the 5 and 3 ends of the RNA fragments, in contrast to Illumina TruSeq protocols, which also include substantial internal biases due to random hexamer priming7,32. The library preparation and correction methods are ordered from the lowest to highest deviation between the median CPM (white point within the violin) and the expected CPM. . 7A) and in obtaining expected log10CPM values (median closest to the red line) with smaller variance across the measured miRNA CPM values (shorter distance between the two ends of the violin plot; Fig. The resulting transcript forms an RNA aptamer that binds . Bazzini, A. The degree of computational correction that can be attained for TGIRT-seq is possible because sequences biases are almost entirely confined to the first three nucleotides from either end of the RNA template. This method uses the ability of TGIRT enzymes to template-switch directly from an artificial RNA template/DNA primer substrate containing an RNA-seq adapter sequence to the 3 end of an RNA template, thereby coupling RNA-seq adapter addition to the initiation of cDNA synthesis6. Additionally, using a miRNA reference set containing an equimolar mixture of 962 human miRNAs, we systematically analyzed 5- and 3-end biases in TGIRT-seq, and developed biochemical and computational methods for ameliorating these biases. 9). R18 was isolated from a random sequence pool by in vitro evolution and stepwise engineering of the initial class I ligase ribozyme (8-10). The libraries were sequenced on an Illumina NextSeq 500 to obtain 1016 million 275-nt paired-end reads, which were mapped to the 962 reference miRNA sequences. 4C,D). A random forest regression model (R2=0.81) based on the first three 5- and 3-end positions was trained on the 962 miRNAs in the combined datasets for the 3 technical replicates obtained using the NTT adapter, and the predicted measurement errors (log10CPM predicted by the model) were plotted against the observed measurement errors (log10CPM obtained directly from sequencing data) for each miRNA. Sources: The expression of genes into proteins and is a process involving two stages called transcription and translation. RNA. S7). Factors other than end biases that may contribute to measurement errors in miRNA representation in TGIRT-seq. Get the most important science stories of the day, free in your inbox. Libraries prepared using each adapter were constructed in triplicate, with the libraries constructed using the NTT adapter requiring 1 round of 1.4X AMPure beads clean-up prior to sequencing compared to 4 rounds for those constructed using the NTC adapter (Supplementary TableS2). For this analysis, we defined over- and under-represented miRNAs as those whose log10 Counts-Per-Million (CPM) values were 1 standard deviation higher and lower, respectively, than the mean log10 CPM for all of the miRNAs in the reference set (Supplementary Fig. Further, TGIRT-seq with the NTT or NTC adapters with computational correction (denoted NTTc and NTCc, respectively) performed slightly better than the 4N protocols in overall sampling bias and variance, and substantially better than commercial small RNA sequencing methods, including NEXTflex, TruSeq, CleanTag, and NEBNext (Fig.
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