3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer

<p>Abstract</p> <p>Background</p> <p>Massive parallel sequencing has the potential to replace microarrays as the method for transcriptome profiling. Currently there are two protocols: full-length RNA sequencing (RNA-SEQ) and 3'-tag digital gene expression (DGE). In this preliminary effort, we evaluated the 3' DGE approach using two reference RNA samples from the MicroArray Quality Control Consortium (MAQC).</p> <p>Results</p> <p>Using Brain RNA sample from multiple runs, we demonstrated that the transcript profiles from 3' DGE were highly reproducible between technical and biological replicates from libraries constructed by the same lab and even by different labs, and between two generations of Illumina's Genome Analyzers. Approximately 65% of all sequence reads mapped to mitochondrial genes, ribosomal RNAs, and canonical transcripts. The expression profiles of brain RNA and universal human reference RNA were compared which demonstrated that DGE was also highly quantitative with excellent correlation of differential expression with quantitative real-time PCR. Furthermore, one lane of 3' DGE sequencing, using the current sequencing chemistry and image processing software, had wider dynamic range for transcriptome profiling and was able to detect lower expressed genes which are normally below the detection threshold of microarrays.</p> <p>Conclusion</p> <p>3' tag DGE profiling with massive parallel sequencing achieved high sensitivity and reproducibility for transcriptome profiling. Although it lacks the ability of detecting alternative splicing events compared to RNA-SEQ, it is much more affordable and clearly out-performed microarrays (Affymetrix) in detecting lower abundant transcripts.</p

4D cardiovascular magnetic resonance velocity mapping of alterations of right heart flow patterns and main pulmonary artery hemodynamics in tetralogy of Fallot

<p>Abstract</p> <p>Background</p> <p>To assess changes in right heart flow and pulmonary artery hemodynamics in patients with repaired Tetralogy of Fallot (rTOF) we used whole heart, four dimensional (4D) velocity mapping (VM) cardiovascular magnetic resonance (CMR).</p> <p>Methods</p> <p>CMR studies were performed in 11 subjects with rTOF (5M/6F; 20.1 ± 12.4 years) and 10 normal volunteers (6M/4F; 34.2 ± 13.4 years) on clinical 1.5T and 3.0T MR scanners. 4D VM-CMR was performed using PC VIPR (Phase Contrast Vastly undersampled Isotropic Projection Reconstruction). Interactive streamline and particle trace visualizations of the superior and inferior vena cava (IVC and SVC, respectively), right atrium (RA), right ventricle (RV), and pulmonary artery (PA) were generated and reviewed by three experienced readers. Main PA net flow, retrograde flow, peak flow, time-to-peak flow, peak acceleration, resistance index and mean wall shear stress were quantified. Differences in flow patterns between the two groups were tested using Fisher's exact test. Differences in quantitative parameters were analyzed with the Kruskal-Wallis rank sum test.</p> <p>Results</p> <p>4D VM-CMR was successfully performed in all volunteers and subjects with TOF. Right heart flow patterns in rTOF subjects were characterized by (a) greater SVC/IVC flow during diastole than systole, (b) increased vortical flow patterns in the RA and in the RV during diastole, and (c) increased helical or vortical flow features in the PA's. Differences in main PA retrograde flow, resistance index, peak flow, time-to-peak flow, peak acceleration and mean wall shear stress were statistically significant.</p> <p>Conclusions</p> <p>Whole heart 4D VM-CMR with PC VIPR enables detection of both normal and abnormal right heart flow patterns, which may allow for comprehensive studies to evaluate interdependencies of post-surgically altered geometries and hemodynamics.</p

CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy–associated TCF4 triplet repeat

PURPOSE: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1). METHODS: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis. RESULTS: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically. CONCLUSION: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease