3′-End Sequencing for Expression Quantification (3SEQ) from Archival Tumor Samples

Gene expression microarrays are the most widely used technique for genome-wide expression profiling. However, microarrays do not perform well on formalin fixed paraffin embedded tissue (FFPET). Consequently, microarrays cannot be effectively utilized to perform gene expression profiling on the vast majority of archival tumor samples. To address this limitation of gene expression microarrays, we designed a novel procedure (3′-end sequencing for expression quantification (3SEQ)) for gene expression profiling from FFPET using next-generation sequencing. We performed gene expression profiling by 3SEQ and microarray on both frozen tissue and FFPET from two soft tissue tumors (desmoid type fibromatosis (DTF) and solitary fibrous tumor (SFT)) (total n = 23 samples, which were each profiled by at least one of the four platform-tissue preparation combinations). Analysis of 3SEQ data revealed many genes differentially expressed between the tumor types (FDR<0.01) on both the frozen tissue (∼9.6K genes) and FFPET (∼8.1K genes). Analysis of microarray data from frozen tissue revealed fewer differentially expressed genes (∼4.64K), and analysis of microarray data on FFPET revealed very few (69) differentially expressed genes. Functional gene set analysis of 3SEQ data from both frozen tissue and FFPET identified biological pathways known to be important in DTF and SFT pathogenesis and suggested several additional candidate oncogenic pathways in these tumors. These findings demonstrate that 3SEQ is an effective technique for gene expression profiling from archival tumor samples and may facilitate significant advances in translational cancer research

Persistent Infection and Promiscuous Recombination of Multiple Genotypes of an RNA Virus within a Single Host Generate Extensive Diversity

Recombination and reassortment of viral genomes are major processes contributing to the creation of new, emerging viruses. These processes are especially significant in long-term persistent infections where multiple viral genotypes co-replicate in a single host, generating abundant genotypic variants, some of which may possess novel host-colonizing and pathogenicity traits. In some plants, successive vegetative propagation of infected tissues and introduction of new genotypes of a virus by vector transmission allows for viral populations to increase in complexity for hundreds of years allowing co-replication and subsequent recombination of the multiple viral genotypes. Using a resequencing microarray, we examined a persistent infection by a Citrus tristeza virus (CTV) complex in citrus, a vegetatively propagated, globally important fruit crop, and found that the complex comprised three major and a number of minor genotypes. Subsequent deep sequencing analysis of the viral population confirmed the presence of the three major CTV genotypes and, in addition, revealed that the minor genotypes consisted of an extraordinarily large number of genetic variants generated by promiscuous recombination between the major genotypes. Further analysis provided evidence that some of the recombinants underwent subsequent divergence, further increasing the genotypic complexity. These data demonstrate that persistent infection of multiple viral genotypes within a host organism is sufficient to drive the large-scale production of viral genetic variants that may evolve into new and emerging viruses

Extensive sequencing of seven human genomes to characterize benchmark reference materials

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly

Replication and recombination of the Red clover necrotic mosaic virus

In this study, Red clover necrotic mosaic virus (RCNMV) was used to better understand the functions of replication proteins and to identify the terminal promoter element involved in viral replication. RCNMV genome contains two positive-sense, single-stranded RNAs. RNA-1 encodes two proteins essential for viral replication: p27 and p88. p88 is a fusion protein containing p27 at its N terminus and RNA dependent RNA polymerase motifs at its C-terminal domain. The function of p27 is not known. In this work, studies of RNA-1 chimerical clones between a highly infectious clone and a poorly infectious clone and subsequent mutagenesis demonstrated that the N-terminal 14 amino acids of p27 and p88 were required for efficient RNA replication. Sequence analysis indicated that it is possibly involved in membrane interaction. Another important aspect of viral replication is template recognition by the replicase at the 3' promoter. The 3' -29 nucleotides of both RCNMV RNA-1 and RNA-2 can be predicted to form an identical stem-loop structure (SLS). Mutational analysis of the SLS indicated that both the structure and the loop sequence were required for viral replication. Within the 5-nt loop region, three discontinuous nucleotides were identified as critical nucleotides for RNA-replicase interaction. The functional groups in these key nucleotides involved in replicase recognition are predicted. The 3' promoter element of RCNMV not only affects viral RNA replication but also influences transgenic recombination. RCNMV RNA-2 encodes a movement protein (MP) that is required for viral cell-to-cell movement and systemic infection. Transgenic Nicotiana benthamiana plants expressing different versions of MP mRNA neither resisted RCNMV nor complemented RNA-1 infection. However, systemic infection was observed in transgenic lines expressing 5' truncated MP mRNA when only RNA-1 was inoculated. Further analysis showed that the infection was resulted from nonhomologous RNA recombination events between infecting RNA-1 and MP transgene mRNA. A replicase-mediated template switch model of the transgenic recombination was proposed. The presence of the 3' promoter element in the transgene mRNA thus was a major factor determining transgenic recombination frequencies. As predicted from the model, transgene mRNA lacking the 3' promoter element would not be a good donor RNA for transgenic recombination. Consequently, no transgenic recombination was detected in transgenic plants expressing the 3' truncated MP mRNA upon inoculation with RCNMV RNA-1

Three discontinuous loop nucleotides in the 3′ terminal stem-loop are required for Red clover necrotic mosaic virus RNA-2 replication

AbstractThe genome of Red clover necrotic mosaic virus (RCNMV) consists of positive-sense, single-stranded RNA-1 and RNA-2. The 29 nucleotides at the 3′ termini of both RNAs are nearly identical and are predicted to form a stable stem-loop (SL) structure, which is required for RCNMV RNA replication. Here we performed a systematic mutagenesis of the RNA-2 3′ SL to identify the nucleotides critical for replication. Infectivity and RNA replication assays indicated that the secondary structure of the 3′ SL and its loop sequence UAUAA were required for RNA replication. Single-nucleotide substitution analyses of the loop further pinpointed three discontinuous nucleotides (L1U, L2A, and L4A) that were vital for RNA replication. A 3-D model of the 3′ SL predicted the existence of a pocket formed by these three nucleotides that could be involved in RNA–protein interaction. The functional groups of the bases participating in this interaction at these positions are discussed

Cutting Performance of Multicomponent AlTiZrN-Coated Cemented Carbide (YG8) Tools during Milling of High-Chromium Cast Iron

In order to improve the cutting performance of cemented-carbide (YG8) tools during the milling of high-chromium cast iron, AlTiZrN coating was deposited on the surface of YG8 samples and milling tools by physical vapor deposition (PVD) technology. The micromorphology and mechanical properties of the coating were studied by the experimental method, and the cutting performance of the coated tools was tested by a milling machining center. The results show that the AlTiZrN coating presents the face-centered cubic (fcc) structure of TiN. The average microhardness is 3887 HV0.05. The bonding strength between the coating and the substrate meets the standard HF3 and is up to the requirements. The coefficient of friction (COF) of the coating is about 0.32. AlTiZrN coating can significantly improve the life of cemented-carbide tools. At cutting speeds of 85, 105, and 125 mm/min, the lives of the AlTiZrN-coated tools increased by 20.7%, 22.4%, and 35.2%, respectively, compared with the uncoated tools. Under the same cutting condition, AlTiZrN-coated tools have better cutting and chip-breaking performance than uncoated tools. With the increase in cutting speed, the workpiece chips produced by AlTiZrN-coated tools are smaller and more uniform, and the scratches on the machined surface are smoother. Therefore, at higher cutting speeds, AlTiZrN-coated tools have more advantages in life and cutting performance than that of uncoated tools. During the cutting process, the wear mechanisms of the AlTiZrN-coated tools mainly included friction, oxidation, and bonding, while oxidation and bonding wear were the main wear mechanisms in the later stage of wear