Transcriptome variation in human tissues revealed by long-read sequencing.

Regulation of transcript structure generates transcript diversity and plays an important role in human disease. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure. In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource.

Familial thrombocytopenia due to a complex structural variant resulting in a WAC-ANKRD26 fusion transcript.

Advances in genome sequencing have resulted in the identification of the causes for numerous rare diseases. However, many cases remain unsolved with standard molecular analyses. We describe a family presenting with a phenotype resembling inherited thrombocytopenia 2 (THC2).

Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms.

Background: Here we present an in-depth characterization of the mechanism of sequencer-induced sample contamination due to the phenomenon of index swapping that impacts Illumina sequencers employing patterned flow cells with Exclusion Amplification (ExAmp) chemistry (HiSeqX, HiSeq4000, and NovaSeq). We also present a remediation method that minimizes the impact of such swaps.

Results: Leveraging data collected over a two-year period, we demonstrate the widespread prevalence of index swapping in patterned flow cell data.

Defining the diverse spectrum of inversions, complex structural variation, and chromothripsis in the morbid human genome.

Background: Structural variation (SV) influences genome organization and contributes to human disease. However, the complete mutational spectrum of SV has not been routinely captured in disease association studies.

Results: We sequenced 689 participants with autism spectrum disorder (ASD) and other developmental abnormalities to construct a genome-wide map of large SV.

Characterizing and measuring bias in sequence data.

Background: DNA sequencing technologies deviate from the ideal uniform distribution of reads. These biases impair scientific and medical applications. Accordingly, we have developed computational methods for discovering, describing and measuring bias.

Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation.

As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data.

High-quality draft assemblies of mammalian genomes from massively parallel sequence data.

Massively parallel DNA sequencing technologies are revolutionizing genomics by making it possible to generate billions of relatively short (~100-base) sequence reads at very low cost. Whereas such data can be readily used for a wide range of biomedical applications, it has proven difficult to use them to generate high-quality de novo genome assemblies of large, repeat-rich vertebrate genomes. To date, the genome assemblies generated from such data have fallen far short of those obtained with the older (but much more expensive) capillary-based sequencing approach.

Targeted exon sequencing by in-solution hybrid selection.

This unit describes a protocol for the targeted enrichment of exons from randomly sheared genomic DNA libraries using an in-solution hybrid selection approach for sequencing on an Illumina Genome Analyzer II. The steps for designing and ordering a hybrid selection oligo pool are reviewed, as are critical steps for performing the preparation and hybrid selection of an Illumina paired-end library. Critical parameters, performance metrics, and analysis workflow are discussed.

Overexpression of BP1, a homeobox gene, is associated with resistance to all-trans retinoic acid in acute promyelocytic leukemia cells.

BP1, a homeobox gene, is overexpressed in the bone marrow of 63% of acute myeloid leukemia patients. In this study, we compared the growth-inhibitory and cyto-differentiating activities of all-trans retinoic acid (ATRA) in NB4 (ATRA-responsive) and R4 (ATRA-resistant) acute promyelocytic leukemia (APL) cells relative to BP1 levels. Expression of two oncogenes, bcl-2 and c-myc, was also assessed.

Evolution of genes and genomes on the Drosophila phylogeny.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution.

Necdin and E2F4 are modulated by rosiglitazone therapy in diabetic human adipose and muscle tissue.

To identify novel pathways mediating molecular mechanisms of thiazolidinediones (TZDs) in humans, we assessed gene expression in adipose and muscle tissue from six subjects with type 2 diabetes before and after 8 weeks of treatment with rosiglitazone. mRNA was analyzed using Total Gene Expression Analysis (TOGA), an automated restriction-based cDNA display method with quantitative analysis of PCR products. The expression of cell cycle regulatory transcription factors E2F4 and the MAGE protein necdin were similarly altered in all subjects after rosiglitazone treatment.

Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1.

Type 2 diabetes mellitus (DM) is characterized by insulin resistance and pancreatic beta cell dysfunction. In high-risk subjects, the earliest detectable abnormality is insulin resistance in skeletal muscle. Impaired insulin-mediated signaling, gene expression, glycogen synthesis, and accumulation of intramyocellular triglycerides have all been linked with insulin resistance, but no specific defect responsible for insulin resistance and DM has been identified in humans.