Data from: A MinION-based pipeline for fast and cost-effective DNA barcoding

DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well-equipped molecular laboratory, is time-consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION™ and demonstrate that one flowcell can generate barcodes for ~500 specimens despite high base-call error rates of MinION™ reads. The pipeline overcomes the errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. Consensus barcodes are overall mismatch-free but retain indel errors that are concentrated in homopolymeric regions. They are addressed with an optional error correction pipeline that uses conserved amino-acid motifs from publicly available barcodes to correct the indel errors. The effectiveness of this pipeline is documented by analysing reads from three MinION™ runs that represent different stages of MinION™ development. They generated data for (1) 511 specimens of a mixed Diptera sample, (2) 575 specimens of ants, and (3) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION barcodes for 490 of the 511 specimens which were assessed against reference Sanger barcodes (N=471). Overall, the MinION barcodes have an accuracy of 99.3%-100% and the number of post-correction ambiguities ranges from 90%). We estimate that up to 1000 barcodes can be generated in one flowcell and that the cost per barcode can be <USD 2

Silencing of miR-34a attenuates cardiac dysfunction in a setting of moderate, but not severe, hypertrophic cardiomyopathy

Therapeutic inhibition of the miR-34 family (miR-34a,-b,-c), or miR-34a alone, have emerged as promising strategies for the treatment of cardiac pathology. However, before advancing these approaches further for potential entry into the clinic, a more comprehensive assessment of the therapeutic potential of inhibiting miR-34a is required for two key reasons. First, miR-34a has ∼40% fewer predicted targets than the miR-34 family. Hence, in cardiac stress settings in which inhibition of miR-34a provides adequate protection, this approach is likely to result in less potential off-target effects. Secondly, silencing of miR-34a alone may be insufficient in settings of established cardiac pathology. We recently demonstrated that inhibition of the miR-34 family, but not miR-34a alone, provided benefit in a chronic model of myocardial infarction. Inhibition of miR-34 also attenuated cardiac remodeling and improved heart function following pressure overload, however, silencing of miR-34a alone was not examined. The aim of this study was to assess whether inhibition of miR-34a could attenuate cardiac remodeling in a mouse model with pre-existing pathological hypertrophy. Mice were subjected to pressure overload via constriction of the transverse aorta for four weeks and echocardiography was performed to confirm left ventricular hypertrophy and systolic dysfunction. After four weeks of pressure overload (before treatment), two distinct groups of animals became apparent: (1) mice with moderate pathology (fractional shortening decreased ∼20%) and (2) mice with severe pathology (fractional shortening decreased ∼37%). Mice were administered locked nucleic acid (LNA)-antimiR-34a or LNA-control with an eight week follow-up. Inhibition of miR-34a in mice with moderate cardiac pathology attenuated atrial enlargement and maintained cardiac function, but had no significant effect on fetal gene expression or cardiac fibrosis. Inhibition of miR-34a in mice with severe pathology provided no therapeutic benefit. Thus, therapies that inhibit miR-34a alone may have limited potential in settings of established cardiac pathology

Therapeutic silencing of miR-652 restores heart function and attenuates adverse remodeling in a setting of established pathological hypertrophy

Expression of microRNA-652 (miR-652) increases in the diseased heart, decreases in a setting of cardioprotection, and is inversely correlated with heart function. The aim of this study was to assess the therapeutic potential of inhibiting miR-652 in a mouse model with established pathological hypertrophy and cardiac dysfunction due to pressure overload. Mice were subjected to a sham operation or transverse aortic constriction (TAC) for 4 wk to induce hypertrophy and cardiac dysfunction, followed by administration of a locked nucleic acid (LNA)-antimiR-652 (miR-652 inhibitor) or LNA control. Cardiac function was assessed before and 8 wk post-treatment. Expression of miR-652 increased in hearts subjected to TAC compared to sham surgery (2.9-fold), and this was suppressed by ∼95% in LNA-antimiR-652-treated TAC mice. Inhibition of miR-652 improved cardiac function in TAC mice (fractional shortening:29±1% at 4 wk post-TAC compared to 35±1% post-treatment) and attenuated cardiac hypertrophy. Improvement in heart function was associated with reduced cardiac fibrosis, less apoptosis and B-type natriuretic peptide gene expression, and preserved angiogenesis. Mechanistically, we identified Jagged1 (a Notch1 ligand) as a novel direct target of miR-652. In summary, these studies provide the first evidence that silencing of miR-652 protects the heart against pathological remodeling and improves heart function.—Bernardo, B. C., Nguyen, S. S., Winbanks, C. E., Gao, X.-M., Boey, E. J. H., Tham, Y. K., Kiriazis, H., Ooi, J. Y. Y., Porrello, E. R., Igoor, S., Thomas, C. J., Gregorevic, P., Lin, R. C. Y., Du, X.-J., McMullen, J. R. Therapeutic silencing of miR-652 restores heart function and attenuates adverse remodeling in a setting of established pathological hypertrophy

Morphological data for control and TAC moderate and severe mice following four weeks of pressure overload and eight weeks after treatment with LNA-control or LNA-antimiR-34a.

<p>BW: body weight, HW: heart weight, AW: atria weight, LW: lung weight, TL: tibia length, HW/TL: heart weight/ tibia length ratio, AW/TL: atria weight/ tibia length ratio, LW/TL: lung weight/ tibia length ratio. Data are shown as mean ± SEM. One-way ANOVA followed by Fisher’s post-hoc test (comparing 5 groups): *P&lt;0.05 vs. control, †P&lt;0.05 vs. control, ‡P&lt;0.001 vs. control, §P&lt;0.05 vs. TAC LNA control of the same group, ∥P&lt;0.05 vs. TAC-moderate of the same treatment group. One-way ANOVA followed by Fisher’s post-hoc test (comparing TAC moderate groups to control, i.e. 3 groups only): #P&lt;0.05 vs. control, **P&lt;0.05 vs. TAC LNA control.</p

Echocardiography data of control and TAC mice at baseline, four weeks post-TAC and eight weeks after treatment with either LNA-control or LNA-antimiR-34a.

<p>BW, body weight; LV, left ventricular; LVPW, LV posterior wall thickness; IVS, interventricular septum thickness; LVEDD, LV end-diastolic dimension; LVESD, LV end-systolic dimension; FS, fractional shortening.</p><p>Data are shown as mean ± SEM. One way ANOVA followed by Fisher’s Post hoc Test.</p><p>*P&lt;0.05 vs. baseline of same group and control at same time point, †P&lt;0.05 vs. TAC LNA control of same group at same time point, ‡P&lt;0.05 vs. same group at 4 weeks post TAC,§P&lt;0.05 vs. TAC moderate of same treatment group at same timepoint, ∥P&lt;0.1 vs. baseline of same group and control at same time point, #P&lt;0.1 vs. TAC LNA control of same group at same time point.</p

Cardiac stress gene expression and fibrosis in moderate and severe models of pressure overload.

<p>(A) Northern blots and quantification of <i>Anp</i>, <i>Bnp</i>, and <i>Serca2a</i> relative to <i>Gapdh</i>, in hearts of control (con), TAC moderate (mod), TAC severe (sev) mice dosed with either LNA-control (c) or LNA-antimiR-34a (a). For Northern blot of <i>Anp</i>, a light exposure (light exp) and dark exposure (dark exp) has been included. N = 3–5 per group. ***P&lt;0.001 vs. control, **P&lt;0.01 vs. control, *P&lt;0.05 vs. control, §P&lt;0.05, †P&lt;0.05, 1 way ANOVA followed by Fisher’s Post Hoc Test (comparing all five groups). When performing 1 way ANOVA followed by Fisher’s Post Hoc Test on control and TAC severe groups only (comparing three groups), N = 3 per group, or control and TAC moderate groups only (comparing three groups), N = 3–5 per group, ∧P&lt;0.05 vs. control. (B) qPCR of <i>β-MHC</i> relative to <i>Hprt1</i> in control, TAC moderate and TAC severe mice dosed with LNA-control or LNA-antimiR-34a. N = 3–5 per group. *P&lt;0.05 vs. control, §P&lt;0.05, ‡P&lt;0.05 vs. control, 1 way ANOVA followed by Fisher’s Post Hoc Test (comparing all 5 groups). (C) LV cross-sections stained with Masson’s trichrome and quantification of LV fibrosis in control, TAC moderate and TAC severe mice dosed with LNA-control or LNA-antimiR-34a. Scale  =  200 µM. N = 4–5 per group. ***P&lt;0.001 vs. control, §P&lt;0.05 vs. TAC-moderate of the same treatment group, 1 way ANOVA followed by Fisher’s Post Hoc Test (comparing all five groups). When comparing control and TAC moderate groups only (comparing three groups), N = 4–5 per group, ∧P&lt;0.05 vs. control, 1 way ANOVA followed by Fisher’s Post Hoc Test.</p

Echocardiography data of control and TAC mice at baseline and four weeks post-TAC.

<p>BW, body weight; LV, left ventricular; LVPW, LV posterior wall thickness; IVS, interventricular septum thickness; LVEDD, LV end-diastolic dimension; LVESD, LV end-systolic dimension; FS, fractional shortening; EF, ejection fraction. Data are shown as mean ± SEM. One way ANOVA followed by Fisher’s Post hoc Test. *P&lt;0.05 vs. baseline of same group and control at same time point, †P&lt;0.05 vs. TAC moderate at same time point.</p

Analysis of miR-34a target gene or protein expression.

<p>(A) Representative Western blots and quantification of VEGF-A and VCL relative to GAPDH in hearts of control (con), TAC moderate (mod), TAC severe (sev) mice dosed with either LNA-control (c) or LNA-antimiR-34a (a). N = 3–5 per group. ∧P&lt;0.05 vs. control when using 1 way ANOVA followed by Fisher’s Post Hoc test on severe group only. (B) qPCR analysis of <i>Sirt1, Sema4b</i>, <i>Vegfb, Pofut1,</i> PNUTS <i>(Pppr10)</i>, <i>Notch 1</i> and Cyclin D1 <i>(Ccnd1)</i> relative to <i>Hprt1</i>. N = 3–5 per group. ∧P&lt;0.05 vs. control when using 1 way ANOVA followed by Fisher’s Post Hoc test on control and TAC severe groups only (comparing three groups).</p

Mice with moderate or severe cardiac dysfunction following four weeks of pressure overload.

<p>Quantification of (A) fractional shortening, (B) ejection fraction and (C) left ventricular posterior wall thickness (LVPW) at four weeks post-TAC in control (CON), TAC moderate (TAC mod) and TAC severe (TAC sev) mice. Scatter plots demonstrate no overlap of fractional shortening and ejection fraction between TAC moderate and TAC severe groups. (D) Representative M-mode echocardiograms. N = 4–9 per group. *P&lt;0.05 vs. control at four weeks post-TAC. †P&lt;0.05. 1 way ANVOA followed by Fisher’s Post Hoc Test.</p