Methods for comprehensive transcriptome analysis using next-generation sequencing and application in hypertrophic cardiomyopathy

Characterization of the RNA transcriptome by next-generation sequencing can produce an unprecedented yield of information that provides novel biologic insights. I describe four approaches for sequencing different aspects of the transcriptome and provide computational tools to analyze the resulting data. Methods that query the dynamic range of gene expression, low expressing transcripts, micro RNA levels, and start-site usage of transcripts are described

Stable Gene Targeting in Human Cells Using Single-Strand Oligonucleotides with Modified Bases

Recent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of $\sim$0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells

Loss of FHL1 induces an age-dependent skeletal muscle myopathy associated with myofibrillar and intermyofibrillar disorganization in mice

Recent human genetic studies have provided evidences that sporadic or inherited missense mutations in four-and-a-half LIM domain protein 1 (FHL1), resulting in alterations in FHL1 protein expression, are associated with rare congenital myopathies, including reducing body myopathy and Emery–Dreifuss muscular dystrophy. However, it remains to be clarified whether mutations in FHL1 cause skeletal muscle remodeling owing to gain- or loss of FHL1 function. In this study, we used FHL1-null mice lacking global FHL1 expression to evaluate loss-of-function effects on skeletal muscle homeostasis. Histological and functional analyses of soleus, tibialis anterior and sternohyoideus muscles demonstrated that FHL1-null mice develop an age-dependent myopathy associated with myofibrillar and intermyofibrillar (mitochondrial and sarcoplasmic reticulum) disorganization, impaired muscle oxidative capacity and increased autophagic activity. A longitudinal study established decreased survival rates in FHL1-null mice, associated with age-dependent impairment of muscle contractile function and a significantly lower exercise capacity. Analysis of primary myoblasts isolated from FHL1-null muscles demonstrated early muscle fiber differentiation and maturation defects, which could be rescued by re-expression of the FHL1A isoform, highlighting that FHL1A is necessary for proper muscle fiber differentiation and maturation in vitro. Overall, our data show that loss of FHL1 function leads to myopathy in vivo and suggest that loss of function of FHL1 may be one of the mechanisms underlying muscle dystrophy in patients with FHL1 mutations

Barcoding bias in high-throughput multiplex sequencing of miRNA

Second-generation sequencing is gradually becoming the method of choice for miRNA detection and expression profiling. Given the relatively small number of miRNAs and improvements in DNA sequencing technology, studying miRNA expression profiles of multiple samples in a single flow cell lane becomes feasible. Multiplexing strategies require marking each miRNA library with a DNA barcode. Here we report that barcodes introduced through adapter ligation confer significant bias on miRNA expression profiles. This bias is much higher than the expected Poisson noise and masks significant expression differences between miRNA libraries. This bias can be eliminated by adding barcodes during PCR amplification of libraries. The accuracy of miRNA expression measurement in multiplexed experiments becomes a function of sample number

Dynamic Cellular Integration Drives Functional Assembly of the Heart’s Pacemaker Complex

SUMMARY Impulses generated by a multicellular, bioelectric signaling center termed the sinoatrial node (SAN) stimulate the rhythmic contraction of the heart. The SAN consists of a network of electrochemically oscillating pacemaker cells encased in a heterogeneous connective tissue microenvironment. Although the cellular composition of the SAN has been a point of interest for more than a century, the biological processes that drive the tissue-level assembly of the cells within the SAN are unknown. Here, we demonstrate that the SAN’s structural features result from a developmental process during which mesenchymal cells derived from a multipotent progenitor structure, the proepicardium, integrate with and surround pacemaker myocardium. This process actively remodels the forming SAN and is necessary for sustained electrogenic signal generation and propagation. Collectively, these findings provide experimental evidence for how the microenvironmental architecture of the SAN is patterned and demonstrate that proper cellular arrangement is critical for cardiac pacemaker biorhythmicity

Dynamic Cellular Integration Drives Functional Assembly of the Heart's Pacemaker Complex.

Impulses generated by a multicellular, bioelectric signaling center termed the sinoatrial node (SAN) stimulate the rhythmic contraction of the heart. The SAN consists of a network of electrochemically oscillating pacemaker cells encased in a heterogeneous connective tissue microenvironment. Although the cellular composition of the SAN has been a point of interest for more than a century, the biological processes that drive the tissue-level assembly of the cells within the SAN are unknown. Here, we demonstrate that the SAN's structural features result from a developmental process during which mesenchymal cells derived from a multipotent progenitor structure, the proepicardium, integrate with and surround pacemaker myocardium. This process actively remodels the forming SAN and is necessary for sustained electrogenic signal generation and propagation. Collectively, these findings provide experimental evidence for how the microenvironmental architecture of the SAN is patterned and demonstrate that proper cellular arrangement is critical for cardiac pacemaker biorhythmicity

Chemically-modified base analogs.

<p>(a–b)Modified base-containing oligos complementary to the non-transcribed strand’s (a)first potential start codon TTG and (b) second potential start codon AAG, where each mismatched base X in the targeting oligo is shown in parenthesis. (c–d) RNAi targeting key mismatch repair proteins MLH1 and MSH2 for the (a)TTG and (b)AAG start codon targeted by oligos containing modified bases. Data was normalized relative to scr shRNA, thus in (c) each MMR component silencing produced a 2-fold improvement for the natural T base, while the improvement for FU was reduced. This is seen to a lesser degree in (d) comparing A and AM, while FA was further improved, suggesting it is more strongly recognized by MMR. n = 4.</p

Effects of PTO in long-term survival of corrected cells.

<p>(a) Varying numbers of PTO bonds 3′ to the mismatch, shown in gray, suggest 3-5PTO bonds as optimal for balancing toxicity and stable targeting frequencies, assayed 14-days after oligo transfection by flow cytometry. (b) Testing the position of PTO bonds, strand targeted and use of modified bases in long term survival. F5-20 used as non correcting control, F5-17, -29 and -30 are complementary to the non-transcribed strand, while F5-31, -32 are complementary to the transcribed. n = 4.</p

Oligonucleotides used in this study.

<p>Sequences shown 5′ to 3′. PTO bonds shown as asterisks (*). dU = deoxyUridine, Fu = 2′-Fluorouracil, Fa = 2′-Fluoroadenine, Am = 2-Aminopurine, mC = 5-Methyl deoxyCytidine.</p

Induction of immune-related genes in oligo-transfected cells.

<p>(a)RT-qPCR relative quantification was performed for key immune-related genes, normalizing oligo-transfected to untransfected cells. (b) Mean Fluorescence Intensity (MFI) of total and EGFP+cells transfected with both F5-17 and F5-38 (Cy5-labeled) oligo at varying concentrations. Transfections were done in 24-well plates. n = 4 (c) Methylation of the CpG sequence present in the targeting oligo has no effect on the %EGFP+ cells. (d) Small-molecule inhibitors against key immune effectors, with DMSO as control, added 24 h after F5-17 oligo transfection assayed for EGFP+ cells at 48 h and 96 h. n = 4.</p