Identification of biological processes and potential inhibitors for aging skin

Aging is a critical risk factor for developing many diseases such as skin diseases. Aging skin is caused by the decline of regenerative potential and function of tissues. Here, we aim to discover the biological function and pathways of the skin from aged people. The GSE39170 dataset was originally created by the Illumina Genome Analyzer II (Homo sapiens). The biological pathways were analyzed by the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), Gene Ontology (GO), and Reactome. KEGG and GO results showed the extracellular matrices (ECMs) were mostly affected in the aging skin. Moreover, we discovered the top ten interacting proteins including FBN1, SPARC, THBS1, DCN, COL1A2, VCAN, LOX, SERPING1, FSTL1, and FBLN5 were involved in the aging skin. Further, we predicted several inhibitors that had the ability to block the aging process by L1000fwd analysis. Thus, this study provides further insights into the mechanism of aging skin.

MicroRNA expression profiles from HEK293 cells expressing H5N1 avian influenza virus non-structural protein 1

H5N1 avian influenza poses a serious threat to the poultry industry and human health. Non-structural protein 1 (NS1) plays an important role in the replication and pathogenesis of avian influenza virus (AIV). However, the function of the NS1 gene is still unclear. In this study, illumina genome analyzer iix screening was used to identify the differentially expressed microRNAs (miRNAs) in HEK293 cells expressing H5N1 AIV NS1. There were 13 differentially expressed miRNAs (hsa-miR-17-5p, hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-31-5p, hsa-miR-20a-5p, hsa-miR-222-3p, hsa-miR-24-3p, hsa-miR-3613-3p, hsa-miR-3178, hsa-miR-4505, hsa-miR-345-3p, hsa-miR-3648, and hsa-miR-455-3p) ( P < 0.01). The qRT-PCR validation results demonstrated that hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-20a-5p, and hsa-miR-3613-3p were upregulated, while hsa-miR-3178 and hsa-miR-4505 were down-regulated. The softwares targetscan and miranda were further used to predict their target genes, and the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results showed that 20 GO terms and 20 KEGG pathways were significantly enriched. Our findings are the first to report expression profiling of miRNA and their functions in H5N1 AIV NS1-expressing HEK293 cells, and pave the way to further elucidating the accurate interaction mechanism between NS1 and virus replication, thus providing brand new insight into the prophylaxis and treatment of H5N1 AIV.

Exploratory analysis and error modeling of a sequencing technology

Next generation DNA sequencing methods have created an unprecedented leap in sequence data generation, thus novel computational tools and statistical models are required to optimize and assess the resulting data. In this report, we explore underlying causes of error for the Illumina Genome Analyzer (IGA) sequencing technology and attempt to quantify their effects using a human bacterial artificial chromosome sequenced to 60,000 fold coverage. Seven potential error predictors are considered: Phred score, read entropy, tile coordinates, local tile density, base position within read, nucleotide call, and lane. With these parameters, logistic regression and log-linear models are constructed and used to show that each of the potential predictors contributes to error (P<1x10-4). With this additional information, we apply the logistic model and achieve a 3% improvement in both the sensitivity and specificity to detect IGA errors. Further, we demonstrate that these modeling approaches can be used as a feedback loop to inform laboratory methods and identify specific machine or run bias.

Mutational Spectrum Of Adult T-ALL

Introduction T-cell acute lymphoblastic leukemia (T-ALL) in adults represents a disease with a rather unfavourable prognosis. Despite the fact that treatment stratification and minimal residual disease (MRD) monitoring have improved survival, there is still need to improve outcome by the development of novel targeted therapies. Therefore, molecular alterations are in the focus of on-going research. Until recently only few candidates were identified as recurringly mutated genes including NOTCH1, FBXW7, PTEN. The development of next generation sequencing (NGS) significantly enlarged this spectrum and identified alterations in additional genes (BCL11B, PHF6, DNM2, CNOT3, KRAS, NRAS, DNMT3A). Whereas a number of putative driver mutations have been characterized, the spectrum of recurring alterations in larger cohorts and their relevance in different leukemic subgroups remains unexplored. To unravel relevant recurring alterations in a large cohort of adult T-ALL and to explore potential target genes for novel therapeutic strategies, we performed targeted high throughput NGS of 88 candidates in 81 T-ALL samples. Patients and methods We investigated 67 adult T-ALL patients enrolled in the trial 07/2003 of the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL). In addition, 14 patients with early T-precursor ALL (ETP-ALL) from other GMALL trials were analysed. Customized biotinylated RNA oligo pools (SureSelect, Agilent) were used to select the targeted regions. We performed 76-bp paired-end sequencing on an Illumina Genome Analyzer IIx platform and reads were mapped to NCBI hg19 RefSeq. For a variant call we required at least a read depth of 20, an allele frequency of 20% and an average base calling quality of Q13. Polymorphisms annotated in dbSNP 135 were excluded. The targeted region comprised 88 genes known to be frequently mutated in ALL, acute myeloid leukemia, myelodysplastic syndrome as well as genes associated with epigenetic regulation, splicing, DNA mismatch repair, and the NOTCHpathway. Results We obtained an average of 1.2 Mbp sequence for each sample, resulting in an average coverage of 120 reads for the target region. 79% of the targeted region was covered with a minimum of 20 reads. After exclusion of polymorphism annotated in dbSNP135, 473 single nucleotide variations (SNV) and small indels were identified, 294 of those resulted in changes on the protein level. On average three (3.1) genes per patient were mutated, and 66 (77%) of the 88 genes were mutated in at least one patient. As expected, the highest mutation rate with 53% was found for NOTCH1, with a higher frequency in thymic T-ALL (67.5%) than in early T-ALL (33.3%). Mutation frequencies of FBXW7 (10%), WT1 (10%), JAK3 (12%), and BCL11B (10%) were in the range of reported frequencies. Recently identified novel alterations in DNM2 (17%), PHF6 (11%), DNMT3A (5%) or RELN (5%) were confirmed in our cohort. Interestingly, genes that had not been described in T-ALL included recurring mutations in the histone methyl-transferase MLL2 (11%), frequently mutated in B-cell lymphomas. Like in lymphoma and in the Kabuki syndrome, MLL2 mutations were distributed over the entire gene without any obvious hot spot region. Also the protocadherins FAT1 (15%) and FAT3 (12%) were recurringly altered. FAT1and its inactivation by mutations were recently linked to activation of the WNT pathway in solid tumours. Affected pathways significantly differed in leukemic subgroups: whereas mutations involving the NOTCH pathway were predominately enriched in the thymic subgroup (75%) and less relevant in early T-ALL (33%, P=0.004), chromatin modifiying genes (17% vs. 5%, P=0.22) and signalling genes (42% vs. 15%, P=0.09) were more frequently mutated in early T-ALL. Spliceosome mutations described in myeloid and mature lymphoid malignancies were present only in a minority (7.4%) of T-ALL. Conclusion Adult T-ALL reveals a highly heterogeneous spectrum of candidate gene mutations. Here we provide an original and comprehensive overview of recurring mutations that unravel preferentially pathways altered in specific leukemic subgroups. In addition, we identified novel candidate genes with potential therapeutic implications (FAT1, EZH2, DNMT3A). These mutations have to be validated in a larger cohort with a focus on clinical implications accompanied by functional assays regarding their use as therapeutic targets. Disclosures: Krebs: Illumina: Honoraria. Greif:Illumina: Honoraria.

First Report of a Tospovirus in Mulberry

Mulberry (Morus alba L.) is an economically important crop grown widely throughout Asia. Various virus-like symptoms including mosaics, vein banding, and chlorotic ringspots have been observed and reported on mulberry trees in China and Japan for decades. However, the etiology of mulberry viral diseases is generally understudied, although two mulberry-infecting viruses, Mulberry latent virus (genus Carlavirus) (2) and Mulberry ringspot virus (genus Nepovirus) (3), have been partially characterized. In a recent (2010 to 2011) field survey in Guangxi Province, China, supported by the local government, the incidence of virus-like diseases of mulberry ranged between 40 and 80%. To identify the viruses infecting mulberry, deep sequencing of small RNAs (4) was conducted using an Illumina Genome Analyzer. Small RNAs were isolated from five samples of mulberry leaves showing various virus-like symptoms and sequenced. Among the contigs assembled, a 445-bp contig (GenBank Accession No. JX268597) was found to share 76.6% nucleotide identity and 83.0% amino acid identity to Groundnut bud necrosis virus (genus Tospovirus, family Bunyaviridae; Accession Nos. U42555 and AAC55521). To obtain a longer cDNA fragment of this virus, a reverse transcription (RT)-PCR was done with primers MV-N-F (5′-AAGCCATCAATGTGCCTCCGGA-3′) and MV-N-R (5′-AACACCATGTCTACCGTCCGTC-3′) that align to the S-RNA sequence encompassing the nucleocapsid (N) gene and a portion of the intergenic region (IGR) of the Tospovirus. PCR products of about 1,000 bp were successfully amplified from the total RNA of the three mulberry samples (sl-1, xcsy-1, and xcsy-4) showing vein banding symptoms, but not from asymptomatic mulberry (jk-1). These PCR products were cloned and sequenced. The lengths of the amplicons were 1,027 bp (isolate sl-1, JX173786), 987 bp (isolate xcsy-1, JX173787), and 979 bp (isolate xcsy-4, JX173788) and the partial IGRs of the sl-1, xcsy-1, and xcsy-4 isolates were 187 bp, 147 bp, and 139 bp, respectively. The coding regions for the N protein were 831 bp and the deduced proteins of 277 amino acid residues were 100% identical for all three isolates. Since the N protein of this virus shared up to only 74.4% identity to other tospoviruses (74.4% to Capsicum chlorosis virus, ABB83818; and 71.5% to Watermelon bud necrosis virus, ABY79095), it may represent a new member of the Tospovirus genus, temporarily named Mulberry vein banding virus (MuVBV), according to the species demarcation criteria for the Bunyaviridae (1). To the best of our knowledge, this is the first report of a Tospovirus infecting M. alba. In an RT-PCR screening of 48 randomly selected mulberry samples suspected to be virus-infected, 32 were MuVBV-positive. Giving the high incidence and the high yield loss associated with Tospovirus and the presence of thrips, suspected vectors for the virus, MuVBV may represent a substantial threat to the silkworm industry in China. References: (1) M. Q. K. Andrew et al. Virus Taxonomy: 9th Report of the ICTV. Elsevier Academic Press, San Diego, 2012. (2) T. Tsuchizaki. Annu. Phytopath. Soc. Japan 42:304, 1976. (3) T. Tsuchizaki et al. Annu. Phytopath. Soc. Japan 37:266, 1971. (4) Q. Wu et al. PNAS. 107:1606, 2010.

Local Assemblies of Paired-End Reduced Representation Libraries Sequenced with the Illumina Genome Analyzer in Maize

The use of next-generation DNA sequencing technologies has greatly facilitated reference-guided variant detection in complex plant genomes. However, complications may arise when regions adjacent to a read of interest are used for marker assay development, or when reference sequences are incomplete, as short reads alone may not be long enough to ascertain their uniqueness. Here, the possibility of generating longer sequences in discrete regions of the large and complex genome of maize is demonstrated, using a modified version of a paired-end RAD library construction strategy. Reads are generated from DNA fragments first digested with a methylation-sensitive restriction endonuclease, sheared, enriched with biotin and a selective PCR amplification step, and then sequenced at both ends. Sequences are locally assembled into contigs by subgrouping pairs based on the identity of the read anchored by the restriction site. This strategy applied to two maize inbred lines (B14 and B73) generated 183,609 and 129,018 contigs, respectively, out of which at least 76% were >200 bps in length. A subset of putative single nucleotide polymorphisms from contigs aligning to the B73 reference genome with at least one mismatch was resequenced, and 90% of those in B14 were confirmed, indicating that this method is a potent approach for variant detection and marker development in species with complex genomes or lacking extensive reference sequences.

Self-organizing Approach for the Human Gut Meta-genome

We extend the self-organizing approach for annotation of a bacterial genome to analyzing the raw sequencing data of the human gut metagenome without sequence assembling. The original approach divides the genomic sequence of a bacterium into non-overlapping segments of equal length and assigns to each segment one of seven ‘phases’, among which one is for the noncoding regions, three for the direct coding regions to indicate the three possible codon positions of the segment starting site, and three for the reverse coding regions. The noncoding phase and the six coding phases are described by two frequency tables of the 64 triplet types or ‘codon usages’. A set of codon usages can be used to update the phase assignment and vice versa. After an initialization of phase assignment or codon usage tables, an iteration leads to a convergent phase assignment to give an annotation of the genome. In the extension of the approach to a metagenome, we consider a mixture model of a number of categories of genomes. The Illumina Genome Analyzer sequencing data of the total DNA from faecal samples are then examined to understand the diversity of the human gut microbiome.

Evaluation of gene expression by RNA-seq after single dose of trastuzumab (T) reveals predictors of pathologic complete response (pCR) in HER2-positive early breast cancer.

10558 Background: The use of a ‘brief exposure’ to single agent T allows the measurement of dynamic changes in the transcriptome that may predict response to T-based combinations. We have shown that most gene expression changes in HER2+ tumors treated with T occur in tumors that ultimately achieve a pCR. Our further analysis suggests several patterns of transcriptional change in pCR tumors suggesting different mechanisms of action of T. RNA-seq analysis provides more in-depth annotation of these mechanisms. Methods: Fresh tumor core biopsies were taken at a 2 week time point after a single dose of T (8mg/m2) from 80 HER2+ early breast cancer patients enrolled on a clinical trial of T>T+C. Nucleic acids were extracted using Qiagen AllPrep and were analyzed with Illumina HT12v3 Beadchip and Illumina 610 QUAD V1 SNP arrays. RNA was also processed for sequencing using the Ovation RNA-Seq System and paired-end sequenced using an Illumina Genome Analyzer IIxRNA-seq data was analyzed with Tophat/Cufflinks. Network analysis was performed with Metacore. Results: Among pCR tumors, distinct patterns of differential expression pre/post T were observed, in both microarray and RNA-seq data. ERBB2 down-regulation was characteristic of pCR in one subgroup by microarray. In this group, differentially expressed genes belonged to interaction networks involved in apoptosis and cell cycle regulation. In contrast, tumors with no change in ErbB2 showed differentially expressed genes that belonged to networks related to chromatin assembly and regulation of immune pathways. NOLC1, RPL41, ZCHHC17, and B2M had altered alternative splicing product distributions in both groups. In the ERBB2 down-regulated group, genes with changed expression were enriched for targets of STAT3 and YY1. Conclusions: RNA-seq and microarray reveal distinct responses in tumors that achieve pCR to T-containing regimens. These methods provide predictive markers for validation in subsequent clinical trials.

195 ALTERED mRNA TRANSCRIPT EXPRESSION OF IN VITRO- VERSUS IN VIVO-MATURED PORCINE OOCYTES

In contrast to oocytes matured in vitro, porcine embryos that result from in vivo maturation and fertilization have a high developmental competence and readily make the transition from oocyte to blastocyst. This observation led us to investigate the transcript profile differences between in vivo- and in vitro-matured porcine oocytes. For the in vivo-matured group, oviducts of 3 gilts of similar genetic background were flushed 2 days after detection of standing oestrus. MII oocytes were collected in pools of 10 and snap frozen in liquid nitrogen for RNA isolation. The in vitro-matured oocytes were obtained by euthanizing 3 gilts, again with a similar genetic background and recovering the ovaries. Follicles (2 to 8 mm in size) were aspirated and oocytes with multiple layers of cumulus cells and uniform cytoplasm were placed in M-199 supplemented with LH, FSH and epidermal growth factor for 42 h. Upon maturation, cumulus cells were stripped and the healthy MII oocytes were collected in pools of 10 and snap frozen. Total RNA was extracted from 3 pools of 10 oocytes for both treatments using an All prep DNA/RNA micro isolation kit (Qiagen, Valencia, CA, USA). Complementary DNA was synthesized using oligo (dT′) primed reverse transcriptase with superscript III (Invitrogen, Carlsbad, CA, USA). Second-strand cDNA was synthesized using DNA polymerase I and sequenced using Illumina Genome Analyzer II. All reads were aligned to a custom-built porcine transcriptome. There were over 18 million reads in the 2 maturation groups that tiled to the 34 433-member transcriptome: 1317 transcripts were detected with a P ≤ 0.1 (Students t-test), a minimum of 7 reads in at least 1 of the treatments and ≥2-fold difference. Real-time PCR was used on selected transcripts. Comparative CT Method was used on an IQ real-time PCR system with the Bio–Rad SYBR green mix. Statistical differences were determined using the Proc general linear model procedure of SAS (SAS Institute Inc., Cary, NC) and means separated with a l.s.d. (P ≤ 0.05). The misrepresented transcripts from the sequencing data were also characterized using the functional annotation tool DAVID. Twelve pathways were overrepresented in the in vitro-matured oocytes (the top 4 are pathways to cancer, spliceosome, cell cycle and ubiquitin-mediated proteolysis). Eight pathways were underrepresented in the in vitro-matured oocytes (the top 4 are cytoskeleton regulation, T-cell receptor signaling pathway, ubiquitin-mediated proteolysis and cell cycle). Eight transcripts were selected for real-time PCR. ZP2 was higher in the in vitro-matured oocytes as determined by both sequencing and real time. ATG4, HSP90, UBAP2 and SOX4 were not different, regardless of assay. SLC7A3, MRPS36 and PDHX2 were not different based on sequencing, but based on real-time MRPS36 and PDHX2, were higher in the in vivo group and SLC7A3 was higher in the in vitro group. In conclusion, there is an abundance of misregulated transcripts and altered pathways in in vitro-matured oocytes. This dataset is a tool that may provide clues to improve the in vitro maturation process so that in vitro-matured oocytes will be more like their in vivo-matured counterparts, thus improving developmental competence. Funded by Food for the 21st Century.