Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments

Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positive rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution

Methylome-wide Analysis of Chronic HIV Infection Reveals Five-Year Increase in Biological Age and Epigenetic Targeting of HLA

HIV-infected individuals are living longer on antiretro-viral therapy, but many patients display signs that in some ways resemble premature aging. To investigate and quantify the impact of chronic HIV infection on aging, we report a global analysis of the whole-blood DNA methylomes of 137 HIV+ individuals under sustained therapy along with 44 matched HIV- individuals. First,we develop and validate epigenetic models of aging that are independent of blood cell composition. Using these models, we find that both chronic and recent HIV infection lead to an average aging advancement of 4.9 years, increasing expected mortality risk by 19%. In addition, sustained infection results in global deregulation of the methylome across \u3e80,000 CpGs and specific hypomethylation of the region encoding the human leukocyte antigen locus (HLA).We find that decreased HLA methylation is predictive of lower CD4/CD8T cell ratio, linking molecular aging, epigenetic regulation, and disease progression

Optimizing sequencing protocols for leaderboard metagenomics by combining long and short reads.

As metagenomic studies move to increasing numbers of samples, communities like the human gut may benefit more from the assembly of abundant microbes in many samples, rather than the exhaustive assembly of fewer samples. We term this approach leaderboard metagenome sequencing. To explore protocol optimization for leaderboard metagenomics in real samples, we introduce a benchmark of library prep and sequencing using internal references generated by synthetic long-read technology, allowing us to evaluate high-throughput library preparation methods against gold-standard reference genomes derived from the samples themselves. We introduce a low-cost protocol for high-throughput library preparation and sequencing

Correction: Role of MicroRNA 1207-5P and Its Host Gene, the Long Non-Coding RNA Pvt1, as Mediators of Extracellular Matrix Accumulation in the Kidney: Implications for Diabetic Nephropathy.

[This corrects the article DOI: 10.1371/journal.pone.0077468.]

<i>PVT1</i>-derived miRNAs.

<p>A. Location of the validated <i>PVT1</i>-derived miRNAs. The grey bars represent the nine <i>PVT1</i> exons. Panels <b>B</b>, <b>C</b> and <b>D</b>, relative quantification of <i>PVT1</i>-derived miRNAs in human renal proximal tubule epithelial cells (RPTEC), podocytes, and mesangial cells (MC) by TaqMan qPCR, respectively. RNU6B and RNU44 were used as endogenous controls. <b>E</b>. Relative quantification of miRNAs in MC grown for 48 h in MsBM medium supplemented with 5% FBS, containing either normal glucose (NG: 5.6 mM), NG +3-O-methyl-D-glucose (3-O-MG) to control for osmotic effects (OC: 5.6 mM glucose +24.4 mM 3-O-MG), or high glucose (HG: 30 mM). Results represent average of three independent experiments. Data are means ± S.D. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * <i>P</i><.05; ** <i>P</i><0.01; *** <i>P</i><0.001.</p

Predicted pairing of target region (top) and miR-1207-5p (bottom) according to TargetScan (www.targetscan.org).

<p>A total of 14 miR-1207-5p's target sites are predicted in the four target genes selected: <i>G6PD, PMEPA1, PDPK1, and SMAD7. G6PD</i>, glucose-6-phosphate dehydrogenase; <i>PMEPA1</i>, prostate transmembrane protein, androgen induced 1; <i>PDPK1</i>, 3-phosphoinositide dependent protein kinase-1; <i>SMAD7</i>, SMAD family member 7.</p

Effect of <i>PVT1</i> on TGF-β1, PAI-1 and FN1.

<p>Relative quantification of TGF-β1, PAI-1, and FN1 mRNA (<b>A</b>) or secreted protein (<b>B</b>) in MC with <i>PVT1</i> over-expression. Cells were transfected with plasmid pCMV-PVT1 or empty vector pCMV (NC, negative control) by electroporation using the Neon System. (<b>C</b>) Changes in expression of FN1 mRNA in MC treated with 30 nM <i>PVT1</i> or NC siRNA, in the presence or absence of the TGF-β signaling inhibitor SB431542. NC siRNA comprised of sequence not found in the human genome. MC were transfected using 5 µl Lipofectamine RNAiMax (Life Technologies) per ml of MsBM media. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significantly different from all the others. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001.</p

Validation of putative miR-1207-5p target genes.

<p>miR-1207-5p target genes were identified by TargetScan software and four genes were selected using Pathway Studio software based on potential biological effects relevant to ECM accumulation. The four genes selected, <i>G6PD, PMEPA1, PDPK1, and SMAD7</i>, were validated as putative miR-1207-5p target genes in MC using 25 nM miR-1207-5p or NC mimic (<b>A</b>), and 50 nM anti-miR-1207-5p or negative control (<b>B</b>). Results in A and B represent averages from three independent experiments. Data are means ± SD. About 20,000 human embryonic kidney 293 (HEK293) cells were seeded per well into a white 96-well plate and co-transfected with 100 ng of the indicated 3′UTR luciferase reporter vectors and 30 nM miR-1207-5p or negative control mimic (Dharmacon) using 0.2 µl per well of Lipofectamine 2000 (<b>C</b>). HEK293 cells were also co-transfected with reporter vectors and 50 nM miR-1207-5p inhibitor or negative control (Dharmacon) (<b>D</b>). Luciferase activity was measured 48 h after transfection using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to the corresponding renilla luciferase activity and plotted as a percentage of the control (HEK293 cells co-transfected with plasmid and control mimic or inhibitor). Approximately 70,000 MC/well were seeded in a 12-well plate and transfected with 3 µl Lipofectamine RNAiMAX (Life Technologies) mixed with 30 nM miR-1207-5p or negative control mimic, and 50 nM anti-miR-1207-5p or negative control. About 24 h after transfection, cells were stimulated with high glucose [30 mM] and incubated for another 24 h. Afterwards, cell culture media was removed, and cells were lysed and phosphorylathed Smad3 was quantified using phospho-SMAD3 (ser423/425) Instant One ELISA (eBiosciences), as per manufacturer's instructions (<b>E</b>). Experiments showed in C, D, and E were performed in quadruplicate. The significance is indicated only for samples that are significant different from all the others. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001. A.U.: arbitrary units. <i>G6PD</i>, glucose-6-phosphate dehydrogenase; <i>PMEPA1</i>, prostate transmembrane protein, androgen induced 1; <i>PDPK1</i>, 3-phosphoinositide dependent protein kinase-1; <i>SMAD7</i>, SMAD family member 7; TGF-β, transforming growth factor beta; PAI-1, plasminogen-activator inhibitor 1; MC, mesangial cells; NC, negative control; p-Smad3, phosphorylated Smad3.</p

Role of MicroRNA 1207-5P and Its Host Gene, the Long Non-Coding RNA <i>Pvt1</i>, as Mediators of Extracellular Matrix Accumulation in the Kidney: Implications for Diabetic Nephropathy

<div><p>Diabetic nephropathy is the most common cause of chronic kidney failure and end-stage renal disease in the Western World. One of the major characteristics of this disease is the excessive accumulation of extracellular matrix (ECM) in the kidney glomeruli. While both environmental and genetic determinants are recognized for their role in the development of diabetic nephropathy, epigenetic factors, such as DNA methylation, long non-coding RNAs, and microRNAs, have also recently been found to underlie some of the biological mechanisms, including ECM accumulation, leading to the disease. We previously found that a long non-coding RNA, the plasmacytoma variant translocation 1 (<i>PVT1</i>), increases plasminogen activator inhibitor 1 (PAI-1) and transforming growth factor beta 1 (TGF-β1) in mesangial cells, the two main contributors to ECM accumulation in the glomeruli under hyperglycemic conditions, as well as fibronectin 1 (FN1), a major ECM component. Here, we report that miR-1207-5p, a <i>PVT1</i>-derived microRNA, is abundantly expressed in kidney cells, and is upregulated by glucose and TGF-β1. We also found that like <i>PVT1</i>, miR-1207-5p increases expression of TGF-β1, PAI-1, and FN1 but in a manner that is independent of its host gene. In addition, regulation of miR-1207-5p expression by glucose and TGFβ1 is independent of <i>PVT1</i>. These results provide evidence supporting important roles for miR-1207-5p and its host gene in the complex pathogenesis of diabetic nephropathy.</p></div