Accurate and exact CNV identification from targeted high-throughput sequence data

<p>Abstract</p> <p>Background</p> <p>Massively parallel sequencing of barcoded DNA samples significantly increases screening efficiency for clinically important genes. Short read aligners are well suited to single nucleotide and indel detection. However, methods for CNV detection from targeted enrichment are lacking. We present a method combining coverage with map information for the identification of deletions and duplications in targeted sequence data.</p> <p>Results</p> <p>Sequencing data is first scanned for gains and losses using a comparison of normalized coverage data between samples. CNV calls are confirmed by testing for a signature of sequences that span the CNV breakpoint. With our method, CNVs can be identified regardless of whether breakpoints are within regions targeted for sequencing. For CNVs where at least one breakpoint is within targeted sequence, exact CNV breakpoints can be identified. In a test data set of 96 subjects sequenced across ~1 Mb genomic sequence using multiplexing technology, our method detected mutations as small as 31 bp, predicted quantitative copy count, and had a low false-positive rate.</p> <p>Conclusions</p> <p>Application of this method allows for identification of gains and losses in targeted sequence data, providing comprehensive mutation screening when combined with a short read aligner.</p

Comparison of the Effects of Aerobic Dance to Water Aerobic Training on Maximal Oxygen Consumption

Water aerobics is one mode of exercise that is gaining popularity. Water aerobics allows adults who participate in an exercise program to improve their cardiorespiratory fitness. The purpose of this study was to determine if water aerobic training is as effective as land dance aerobic training in improving cardiorespiratory fitness in sedentary healthy adults. Eighteen subjects participated in this study; nine in dance aerobics and nine in water aerobics. Before and after eight weeks of training, subjects performed a graded maximal exercise test on a Schwinn Air-Dyne while maximal oxygen uptake was measured via a Beckman Metabolic Cart. ANCOVA was used to analyze the data with mode, age, weight, and prior activity level as covariates. There was no significant difference found in the training effects between the water and land aerobics and both groups improved their maximal oxygen uptake significantly (p-value = 0.004). Therefore, water aerobics appears to be an effective means of improving cardiorespiratory fitness in sedentary healthy adults

Sensitive Phenotypic Detection of Minor Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Variants

Detection of drug-resistant variants is important for the clinical management of human immunodeficiency virus type 1 (HIV-1) infection and for studies on the evolution of drug resistance. Here we show that hybrid elements composed of the Saccharomyces cerevisiae retrotransposon Ty1 and the reverse transcriptase (RT) of HIV-1 are useful tools for detecting, monitoring, and isolating drug-resistant reverse transcriptases. This sensitive phenotypic assay is able to detect nonnucleoside reverse transcriptase inhibitor-resistant RT domains derived from mixtures of infectious molecular clones of HIV-1 in plasma and from clinical samples when the variants comprise as little as 0.3 to 1% of the virus population. Our assay can characterize the activities and drug susceptibilities of both known and novel reverse transcriptase variants and should prove useful in studies of the evolution and clinical significance of minor drug-resistant viral variants

Hybrid Capture and Next-Generation Sequencing Identify Viral Integration Sites from Formalin-Fixed, Paraffin-Embedded Tissue

Although next-generation sequencing (NGS) has been the domain of large genome centers, it is quickly becoming more accessible to general pathology laboratories. In addition to finding single-base changes, NGS allows for the detection of larger structural variants, including insertions/deletions, translocations, and viral insertions. We describe the use of targeted NGS on DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue, and show that the short read lengths of NGS are ideally suited to fragmented DNA obtained from FFPE tissue. Further, we describe a novel method for performing hybrid-capture target enrichment using PCR-generated capture probes. As a model, we captured the 5.3-kb Merkel cell polyomavirus (MCPyV) genome in FFPE cases of Merkel cell carcinoma using inexpensive, PCR-derived capture probes, and achieved up to 37,000-fold coverage of the MCPyV genome without prior virus-specific PCR amplification. This depth of coverage made it possible to reproducibly detect viral genome deletions and insertion sites anywhere within the human genome. Out of four cases sequenced, we identified the 5′ insertion sites in four of four cases and the 3′ sites in three of four cases. These findings demonstrate the potential for an inexpensive gene targeting and NGS method that can be easily adapted for use with FFPE tissue to identify large structural rearrangements, opening up the possibility for further discovery from archival tissue