Jorge Gandara

Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study.

High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A–selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.

Geospatial Resolution of Human and Bacterial Diversity with City-Scale Metagenomics

SUMMARY The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station’s history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.

N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection

Summary The RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove this modification, as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A methyltransferases or an m6A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determined that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Altogether, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.

Genome assembly and geospatial phylogenomics of the bed bug Cimex lectularius.

The common bed bug (Cimex lectularius) has been a persistent pest of humans for thousands of years, yet the genetic basis of the bed bugs basic biology and adaptation to dense human environments is largely unknown. Here we report the assembly, annotation and phylogenetic mapping of the 697.9-Mb Cimex lectularius genome, with an N50 of 971 kb, using both long and short read technologies. A RNA-seq time course across all five developmental stages and male and female adults generated 36,985 coding and noncoding gene models. The most pronounced change in gene expression during the life cycle occurs after feeding on human blood and included genes from the Wolbachia endosymbiont, which shows a simultaneous and coordinated host/commensal response to haematophagous activity. These data provide a rich genetic resource for mapping activity and density of C. lectularius across human hosts and cities, which can help track, manage and control bed bug infestations.

Genomic DNA transposition induced by human PGBD5

Transposons are mobile genetic elements that are found in nearly all organisms, including humans. Mobilization of DNA transposons by transposase enzymes can cause genomic rearrangements, but our knowledge of human genes derived from transposases is limited. In this study, we find that the protein encoded by human PGBD5, the most evolutionarily conserved transposable element-derived gene in vertebrates, can induce stereotypical cut-and-paste DNA transposition in human cells. Genomic integration activity of PGBD5 requires distinct aspartic acid residues in its transposase domain, and specific DNA sequences containing inverted terminal repeats with similarity to piggyBac transposons. DNA transposition catalyzed by PGBD5 in human cells occurs genome-wide, with precise transposon excision and preference for insertion at TTAA sites. The apparent conservation of DNA transposition activity by PGBD5 suggests that genomic remodeling contributes to its biological function. DOI: http://dx.doi.org/10.7554/eLife.10565.001

Mining the Archives: A Cross-Platform Analysis of Gene Expression Profiles in Archival Formalin-Fixed Paraffin-Embedded Tissues

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for transcriptomic research. However, use of FFPE samples in genomic studies has been limited by technical challenges resulting from nucleic acid degradation. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues preserved in formalin for different amounts of time using 2 DNA microarray protocols and 2 whole-transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other methods by having the highest correlations of differentially expressed genes (DEGs), and best overlap of pathways, between FRO and FFPE groups. The effect of sample time in formalin (18 h or 3 weeks) on gene expression profiles indicated that test article treatment, not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18 h and 3 week FFPE samples compared with FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of time in paraffin on genomic profiles. Ribo-depletion RNA-seq analysis of 8-, 19-, and 26-year-old control blocks resulted in comparable quality metrics, including expected distributions of mapped reads to exonic, untranslated region, intronic, and ribosomal fractions of the transcriptome. Overall, our results indicate that FFPE samples are appropriate for use in genomic studies in which frozen samples are not available, and that ribo-depletion RNA-seq is the preferred method for this type of analysis in archival and long-aged FFPE samples.

Modern Methods for Delineating Metagenomic Complexity

We appreciate the comments of Ackelsberg et al. (Ackelsberg et al., 2015xAckelsberg, J., Rakeman, J., Hughes, S., Peterson, J., Mead, P., Schriefer, M., Kingry, L., Hoffmaster, A., and Gee, J. Cell Syst. 2015; 1: 4–5Abstract | Full Text | Full Text PDF | Scopus (1)See all ReferencesAckelsberg et al., 2015) and have decided to revise the paper (Afshinnekoo et al., 2015xAfshinnekoo, E., Meydan, C., Chowdhury, S., Jaroudi, D., Boyer, C., Bernstein, N., Maritz, J.M., Reeves, D., Gandara, J., Chhangawala, S. et al. Cell Syst. 2015; 1: 72–87Abstract | Full Text | Full Text PDF | Scopus (23)See all ReferencesAfshinnekoo et al., 2015) as follows:Figure 3B has been corrected to show the general coverage of the Yersinia pestis pMT1 plasmid, but not the murine toxin gene (yMT). The initial claim of “…consistent 20× coverage across the murine toxin gene…” was erroneously based on looking at annotations from related plasmids and comparing different reference sequences. In actuality no reads mapped to the yMT gene.The result of low coverage to the Bacillus anthracis plasmids (pXO1 and pXO2) and no evidence of plcR SNP—an often defining feature of anthrax—is now reported in the Results section.The language in the Summary, Results, and Discussion has been revised, and speculative text about pathogenic organisms has been deleted. We now state that although all our metagenomic analysis tools identified reads with similarity to B. anthracis and Y. pestis sequences, there is minimal coverage to the backbone genome of these organisms, and there is no strong evidence to suggest these organisms are in fact present and no evidence of pathogenicity.Furthermore, in regards to the concerns of the culture methods we have posted subsequent details on the study website (http://www.pathomap.org/2015/04/13/culture-methods/) and below.A second culture experiment was performed to address the question of antibiotic resistance (Afshinnekoo et al., 2015xAfshinnekoo, E., Meydan, C., Chowdhury, S., Jaroudi, D., Boyer, C., Bernstein, N., Maritz, J.M., Reeves, D., Gandara, J., Chhangawala, S. et al. Cell Syst. 2015; 1: 72–87Abstract | Full Text | Full Text PDF | Scopus (23)See all ReferencesAfshinnekoo et al., 2015, Figure 4A). Bacteria were cultured in LB agar and then spread onto LB plates, after lawn growth, single colonies were picked and then plated onto antibiotic plates (kanamycin – 50 ug/ml, chloramphenicol – 35 ug/ml, and ampicillin – 100 ug/ml) and growth was assessed. Plates were incubated at 37°C. As a control, air samples were taken and cultured at every location. In all cases, these did not yield growth. The non-selective plate done last when replica plating also serves as a control. There was no quantitative confirmation of bacterial versus non-bacterial organisms, although there was no observable fungal growth in the samples. Further experiments are being done to dive deeper into the question of viability of microorganisms on the subway system as well as the presence of antibiotic-resistant bacteria.The field of metagenomics is relatively new but has great potential to serve an incredibly important role both in our understanding of the world around us—with key applications in the built environment—as well as the clinical realm. Nevertheless, there are still major hurdles and challenges that the field faces in order to realize this potential. We welcome and appreciate the discussion (http://microbe.net/2015/02/17/the-long-road-from-data-to-wisdom-and-from-dna-to-pathogen/) prompted by our study, and we anticipate that this large dataset will enable further experimentation, additional testing of taxonomic tools, and hopefully help in developing methodologies for metagenomic analysis.

Abstract 1103: Human tumorigenesis induced by endogenous DNA transposase

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Recent cancer genome surveys have revealed extremely low rates of coding gene mutations in distinct tumor subtypes, suggesting that alternative mechanisms must contribute to their pathogenesis. Transposons are mobile genetic elements that are found in all living organisms, including humans where they occupy nearly half of the genome. Their mobilization can cause structural rearrangements in normal and cancer cells. However, it remains unknown whether transposition is a cause of cellular transformation or merely a bystander effect of dysregulated gene expression. Here, we report that PGBD5, a recently characterized human gene related to the piggyBac transposase from the cabbage looper moth, is aberrantly expressed in rhabdoid tumors, medulloblastoma, acute leukemias, and some sarcomas and carcinomas. Ectopic expression of PGBD5 in non-transformed primary human cells is sufficient to induce anchorage independence in vitro and penetrant tumor formation in immunodeficient mice in vivo. PGBD5 expression is sufficient to induce genomic mobilization of engineered DNA transposons in human cells, and purified recombinant PGBD5 exhibits transposase domain-dependent endonuclease activity in vitro. Flanking-sequence exponential anchored PCR and massively parallel sequencing of DNA transposon integrations revealed distinct activity on piggyBac-like inverted terminal repeats, and preference for specific euchromatic human genomic loci. This enables mapping of structural rearrangements of endogenous human transposable elements in primary human tumor genomes, some of which target genes involved in cellular transformation. We find that PGBD5 transposase-induced cell transformation is associated with morphologic de-differentiation, induction of distinct Polycomb gene expression programs and structural chromatin remodeling, consistent with its epigenetic control. These findings reveal an unanticipated mechanism of human tumorigenesis, genomic plasticity and structural alterations of non-coding regulatory genomic loci in human cancer. Citation Format: Anton Henssen, Amy Eisenberg, Eileen Jiang, Elizabeth Henaff, Richard Koche, Melissa Burns, Julianne R. Carson, Gouri Nanjangud, Eric Still, Jorge Gandara, Paolo Cifani, Avantika Dhabaria, Xiaodong Huang, Elisa de Stanchina, Elizabeth Mullen, Hanno Steen, Elizabeth Perlman, Jeffrey Dome, Cristina Antonescu, Cedric Feschotte, Christopher E. Mason, Alex Kentsis. Human tumorigenesis induced by endogenous DNA transposase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1103. doi:10.1158/1538-7445.AM2015-1103