Journal: Scientific Reports

Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

doi: 10.1038/s41598-019-44457-z

Figure Lengend Snippet: TGIRT-seq workflow and design of an improved R2R adapter that decreases adapter-dimer formation. ( A ) TGIRT-seq workflow. 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. For the preparation of TGIRT-seq libraries from pools of RNAs, the DNA primer consists of a mixture of DNA oligonucleotides that leave A, C, G, and T 3′ overhangs (denoted N). After pre-incubation of the TGIRT enzyme with the target RNAs and template-primer (see Methods), template-switching and reverse transcription of an RNA template are initiated by adding dNTPs. 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). A pre-adenylated oligonucleotide containing the reverse complement of an Illumina R1 sequence (R1R) is then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA ligase (New England Biolabs), followed by MinElute clean-up and 12 cycles of PCR amplification with primers that add indices and capture sites for Illumina sequencing. 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. ( B ) Taking into account known biases of the 5′ App DNA/RNA ligase , , , 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. ( C ) Bioanalyzer traces comparing adapter-dimer formation using the previous NTC and improved NTT R2R adapters. 2 pmole of the NTC or NTC R2R adapter was ligated to 40 pmole of adenylated R1R adapter followed by 12 cycles of PCR according to the TGIRT-seq protocol and 1 round of clean-up with 1.4X AMPure beads to remove salt, PCR primers, and adapter dimers. The products were analyzed by using a 2100 Bioanalyzer (Agilent) with a high sensitivity DNA chip. M: internal markers in the NTC (red) or NTT (blue) traces.

Article Snippet: Thermostable group II intron reverse transcriptases (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC.

Techniques: Sequencing, Blocking Assay, Incubation, Reverse Transcription, Amplification, Illumina Sequencing, Modification, Residue

Journal: Scientific Reports

Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

doi: 10.1038/s41598-019-44457-z

Figure Lengend Snippet: Bioanalyzer traces of TGIRT-seq libraries constructed from varying amounts of different-sized RNA oligonucleotides using either the NTC or NTT adapter. TGIRT-seq libraries were prepared from ( A ) 40-nt or ( B ) 20-nt RNA oligonucleotides using the workflow of Fig. . After PCR for 12 cycles and one round of 1.4X AMPure beads clean-up, the libraries were analyzed on a 2100 Bioanalyzer (Agilent) using a high sensitivity DNA chip. M: internal markers.

Article Snippet: Thermostable group II intron reverse transcriptases (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC.

Techniques: Construct

Journal: Scientific Reports

Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

doi: 10.1038/s41598-019-44457-z

Figure Lengend Snippet: TGIRT-seq of ribo-depleted fragmented UHRR with ERCC spike-ins using the NTT and NTC adapters. TGIRT-seq libraries were prepared in triplicate for each adapter and sequenced on an Illumina NextSeq 500 to obtain 58–105 million 75-nt paired-end reads, which were mapped to a human reference genomic (Ensembl GRCh38) modified to include additional rRNA repeats (Methods and Supplementary Table ). The datasets were used to generate stacked bar graphs showing the percentages of: ( A ) read-pairs that mapped concordantly in the annotated orientation to different categories of genomic features; ( B ) small ncRNA reads that mapped to different classes of small ncRNAs; ( C ) protein-coding gene reads that mapped to the sense or antisense strand; ( D ) bases in protein-coding gene reads that mapped to coding sequences (CDS), introns, 5′- and 3′-untranslated regions (UTRs), and intergenic regions. The name of the dataset is indicated below. ( 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).

Article Snippet: Thermostable group II intron reverse transcriptases (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC.

Techniques: Modification

Journal: Scientific Reports

Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

doi: 10.1038/s41598-019-44457-z

Figure Lengend Snippet: 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. TGIRT-seq libraries were prepared from the Miltenyi miRXplore miRNA reference set containing 962 equimolar human miRNAs (Supplementary Table and Methods). Datasets for each method (three combined datasets for NTC, NTT, MTT, and NTT and a single dataset for NTT/6N) were used to plot both the empirical cumulative distribution function (ECDF) of the log 2 median-normalized counts for each miRNA ranked from least to most abundant (left panels), and the abundance-adjusted nucleotide frequencies at the 5′ end (positions +1 to +6) and 3′ end (positions −1 to −6) of the miRNA sequences in the dataset relative to those in the miRNA reference set (middle and right panels). Only uniquely mapped reads were counted. The numbers within the ECDF plots for each method indicate the root-mean-square error (RMSE) for over-represented miRNAs (top right), under-represented miRNAs (bottom left), and all miRNAs (top left). The curve plotted as a dashed line at the bottom of the ECDF plots indicates the distribution density of the 962 miRNAs in the dataset. ( A ) Miltenyi miRXplore reference set showing the ECDF plot layout (left panel) and the aggregate 5′- and 3′-nucleotide frequencies for all miRNAs in the Miltenyi miRXplore reference set assuming equimolar concentrations of the 962 miRNAs. ( B–G ) ECDF plots (left panels) and plots of the abundance-adjusted nucleotide frequencies at the 5′- and 3′- ends of miRNAs in TGIRT-seq datasets relative to those in the miRNA reference set (middle and right panels) for datasets obtained using ( B ) the NTC adapter; ( C ) the NTT adapter; ( D ) a modified NTT adapter mix in which the 3′ A overhang was replaced with a 3′ diaminopurine (denoted MTT); ( E ) a modified NTT adapter mix with an altered ratio of 3′ overhangs (A:C:G:T = 6.6:0.4:1:1; denoted NTTR); ( F ) the NTT adapter used in combination with an R1R adapter with six randomized nucleotides at its 5′ end (denoted NTT/6N); and ( G ) the NTT adapter after computational correction of 5′- and 3′-end biases (denoted NTTc).

Article Snippet: Thermostable group II intron reverse transcriptases (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC.

Techniques: Comparison, Modification

Journal: Scientific Reports

Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

doi: 10.1038/s41598-019-44457-z

Figure Lengend Snippet: Effect of 5′- and 3′-end sequences on the representation of miRNAs in TGIRT-seq datasets. ( A ) Principal component analysis biplot for over- and under-represented miRNAs in TGIRT-seq of the Miltenyi miRXplore miRNA reference set in combined datasets for the three technical replicates obtained using the NTT adapter. The first three bases from the 5′- and 3′ ends of over- and under-represented miRNAs (defined as those whose log 2 CPM was at least one standard deviation higher or lower, respectively, than the mean log 2 CPM for all miRNAs in the reference set; Supplementary Fig. ) were subject to principal component analysis. The first two principal components are shown. Each point indicates a miRNA, with over- and under-represented miRNAs colored as indicated in the Figure. ( B ) Relative importance of features of the first principal component. The fitted values from the first principal component are plotted for each base at each nucleotide position (feature) in ascending order. 5′- and 3′-end nucleotides are color coded as indicated in the Figure. ( C ) Random forest regression modeling of miRNA-seq quantification errors. A random forest regression model ( R 2 = 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 (∆log 10 CPM predicted by the model) were plotted against the observed measurement errors (∆log 10 CPM obtained directly from sequencing data) for each miRNA. The blue line shows the fitted linear regression between the observed and predicted measurement errors, and the red line indicates hypothetical perfect prediction with slope = 1 and y-intercept = 0. ( D ) Relative importance of the position-specific preferences in TGIRT-seq. The relative importance of the 5′- and 3′-end positions from the random forest regression model were plotted in descending order. Each bar represents the relative importance of the indicated position color coded as indicated in the figure.

Article Snippet: Thermostable group II intron reverse transcriptases (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC.

Techniques: Standard Deviation, Sequencing

Journal: Scientific Reports

Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

doi: 10.1038/s41598-019-44457-z

Figure Lengend Snippet: TGIRT-seq of the Miltenyi miRXplore miRNA reference set using R2 RNA/R2R DNA adapters with different ratios of the 3′-DNA overhang nucleotides. 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. The expected ratio in the miRNA reference set is shown by the bar graph at the right. Only uniquely mapped reads were counted.

Article Snippet: Thermostable group II intron reverse transcriptases (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC.

Techniques:

Journal: Scientific Reports

Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

doi: 10.1038/s41598-019-44457-z

Figure Lengend Snippet: 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. For published datasets containing additional miRNAs, in silico subsamples containing only the 962 reference set miRNAs were used for the comparisons. ( A ) RNA-seq saturation curves. The curves show the number of reference set miRNAs with at least 10 reads at bins of 200 reads. As additional reads were included, the number of miRNAs with at least 10 reads increased. Curves were truncated at 3 million reads. The dotted red line at the top indicates the number of miRNAs in the Miltenyi miRXplore reference set. Each curve represents combined datasets, color-coded by the sequencing method as shown in the Figure for the best (4N ligation/NEXTflex; n = 24) and worst (NEBNext; n = 12) methods from the comparison of Giraldez et al . , as well as TGIRT-seq (n = 3 for libraries prepared with the NTT, MTT, and NTC adapters), TGIRT-seq with the NTTR adapter (n = 3), TGIRT-seq with the NTT adapter and an R1R adapter containing six randomized 5′-end positions (NTT/6N; n = 1), and the TGIRT-CircLigase method (n = 1; Mohr et al . ). Other library preparation methods (gray lines) include NEBNext, TruSeq and CleanTag. ( B ) Violin plots of miRNA abundance in datasets obtained by different methods. The plots show the distribution of log 10 CPM for each miRNA in the reference set for each library preparation method (miRNA count = 2,886 for NTTc, 2,885 for NTCc, 23,088 for 4N ligation, 961 for TGIRT-CircLigase, 2,886 for NTTR, 5,522 for NEXTflex, 2,886 for MTT, 2,886 for NTC, 2,886 for NTT, 962 for NTT/6N, 30,757 for TruSeq, 3,815 for CleanTag, and 11,452 for NEBNext). 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. ). The black horizontal line indicates the expected CPM values (CPM = 1,039.5) for each miRNA for a uniform distribution of 1,000,000 reads to 962 miRNAs ( i . e ., unbiased sampling for each miRNA). 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. 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.

Article Snippet: Thermostable group II intron reverse transcriptases (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC.

Techniques: RNA Sequencing, In Silico, Sequencing, Ligation, Comparison, Sampling

Journal: Scientific Reports

Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

doi: 10.1038/s41598-019-44457-z

Figure Lengend Snippet: Representation of the Miltenyi miRXplore miRNA reference set in datasets obtained by TGIRT-seq with the NTT adapter before and after computational correction compared to representation of the same miRNAs in datasets obtained using 4N protocols. ( A ) miRNA representation for TGIRT-seq NTT versus 4N. Log 10 CPM 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 . ). Each point represents one miRNA. ( B ) The same comparison as (A) after computational correction of the TGIRT-seq NTT dataset using the random forest regression model (Fig. ,D). In (B), miRNAs are color-coded by their lengths (scale to the right). The purple dotted lines delineate 95% confidence intervals (2 standard deviations from the mean) of the miRNAs for 4N (vertical dotted lines) or NTT (horizontal dotted lines). The box formed by the intersections of the dotted lines encompasses 892 miRNAs that lie within these confidence intervals and were used for comparison with over- and under-represented miRNAs in Fig. . The expected CPM values (CPM = 1,039.5 for each of the 962 equimolar miRNAs) are indicated by horizontal and vertical orange lines for TGIRT-seq and the 4N protocols, respectively. The diagonal red line indicates cases where the CPM values from NTT are equal to those for 4N protocols. ( C ) Correlation between miRNA abundances and miRNA length. Two-dimensional kernel density estimation of the distribution for miRNA abundances and lengths (n = 962) is shown. The linear regression, with the equation: log 10 CPM = 0.09 (miRNA size) +0.9, is plotted as a red line, and miRNAs with length <21 or >23 nt are indicated as white crosses. The coefficient of determinant ( R 2 ) is indicated in the plot. The color scale indicates the numbers of miRNAs not shown as crosses.

Article Snippet: Thermostable group II intron reverse transcriptases (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC.

Techniques: Comparison

Journal: Scientific Reports

Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

doi: 10.1038/s41598-019-44457-z

Figure Lengend Snippet: Factors other than end biases that may contribute to measurement errors in miRNA representation in TGIRT-seq. The figure shows violin plots comparing several potentially bias-inducing characteristics in over-represented (n = 8) or under-represented miRNAs (n = 27) in combined TGIRT-seq datasets obtained using the NTT adapter defined as those with log 10 CPM values two or more standard deviations higher than the mean log 10 CPM compared to the remaining 927 miRNAs (those within the center box in Fig. ). The characteristics compared include: ( A ) miRNA length; ( B ) GC content; ( C ) the minimum free energy of the most stable predicted secondary structure (self-fold energy) computed by the Vienna RNA package; ( D ) the predicted minimum free energy of base pairing between the R1R adapter and the miRNA cDNA with attached R2R adapter to which it is ligated in the second step of TGIRT-seq (Fig. ) computed by Vienna RNA package (co-fold energy); ( E ) the number of unpaired (free) 3′ nucleotides in the predicted secondary structure; and ( F ) the number of unpaired (free) 5′ nucleotides in the predicted secondary structure. 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).

Article Snippet: Thermostable group II intron reverse transcriptases (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC.

Techniques: