Scott Tighe

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.

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.

mTORC1 Activation Blocks BrafV600E-Induced Growth Arrest but Is Insufficient for Melanoma Formation

Braf(V600E) induces benign, growth-arrested melanocytic nevus development, but also drives melanoma formation. Cdkn2a loss in Braf(V600E) melanocytes in mice results in rare progression to melanoma, but only after stable growth arrest as nevi. Immediate progression to melanoma is prevented by upregulation of miR-99/100, which downregulates mTOR and IGF1R signaling. mTORC1 activation through Stk11 (Lkb1) loss abrogates growth arrest of Braf(V600E) melanocytic nevi, but is insufficient for complete progression to melanoma. Cdkn2a loss is associated with mTORC2 and Akt activation in human and murine melanocytic neoplasms. Simultaneous Cdkn2a and Lkb1 inactivation in Braf(V600E) melanocytes results in activation of both mTORC1 and mTORC2/Akt, inducing rapid melanoma formation in mice. In this model, activation of both mTORC1/2 is required for Braf-induced melanomagenesis.

Real-Time DNA Sequencing in the Antarctic Dry Valleys Using the Oxford Nanopore Sequencer

The ability to sequence DNA outside of the laboratory setting has enabled novel research questions to be addressed in the field in diverse areas, ranging from environmental microbiology to viral epidemics. Here, we demonstrate the application of offline DNA sequencing of environmental samples using a hand-held nanopore sequencer in a remote field location: the McMurdo Dry Valleys, Antarctica. Sequencing was performed using a MK1B MinION sequencer from Oxford Nanopore Technologies (ONT; Oxford, United Kingdom) that was equipped with software to operate without internet connectivity. One-direction (1D) genomic libraries were prepared using portable field techniques on DNA isolated from desiccated microbial mats. By adequately insulating the sequencer and laptop, it was possible to run the sequencing protocol for up to 2½ h under arduous conditions.

Mammary gland morphological and gene expression changes underlying pregnancy protection of breast cancer tumorigenesis

A full-term pregnancy early in life reduces lifetime risk of developing breast cancer, and the effect can be mimicked in rodents by full-term pregnancy or short-term treatment with exogenous estrogen and progesterone. To gain insight into the protective mechanism, 15 3-mo-old postpubertal virgin Lewis rats were randomly assigned to three groups: control (C), pregnancy (P), or hormone (H). The P group animals underwent a full-term pregnancy, and H group animals were implanted subcutaneously with silastic capsules filled with ethynyl estradiol and megesterol acetate for 21 days. C and P animals were implanted with sham capsules. On day 21 capsules were removed, which was followed by a 49-day involution period, euthanasia, and mammary tissue collection. Global gene expression was measured using Rat Genome 230.2 Arrays. Histological analysis revealed that P and H treatments induced sustained morphological changes in the mammary gland with significantly increased percentages of mammary parenchyma and stromal tissues and higher ratio of stroma to parenchyma. Transcriptome analysis showed that P and H treatments induced sustained global changes in gene expression in the mammary gland. Analysis of commonly up- and downregulated genes in P and H relative to C treatment showed increased expression of three matrix metallopeptidases (Mmp3, 8, and 12), more differentiated mammary phenotype, enhanced innate and adaptive immunity, and reduced cell proliferation and angiogenic signatures. The sustained morphological and global gene expression changes in mammary tissue after pregnancy and hormone treatment may function together to provide the protective effect against breast cancer.

Comprehensive benchmarking and ensemble approaches for metagenomic classifiers

BackgroundOne of the main challenges in metagenomics is the identification of microorganisms in clinical and environmental samples. While an extensive and heterogeneous set of computational tools is available to classify microorganisms using whole-genome shotgun sequencing data, comprehensive comparisons of these methods are limited.ResultsIn this study, we use the largest-to-date set of laboratory-generated and simulated controls across 846 species to evaluate the performance of 11 metagenomic classifiers. Tools were characterized on the basis of their ability to identify taxa at the genus, species, and strain levels, quantify relative abundances of taxa, and classify individual reads to the species level. Strikingly, the number of species identified by the 11 tools can differ by over three orders of magnitude on the same datasets. Various strategies can ameliorate taxonomic misclassification, including abundance filtering, ensemble approaches, and tool intersection. Nevertheless, these strategies were often insufficient to completely eliminate false positives from environmental samples, which are especially important where they concern medically relevant species. Overall, pairing tools with different classification strategies (k-mer, alignment, marker) can combine their respective advantages.ConclusionsThis study provides positive and negative controls, titrated standards, and a guide for selecting tools for metagenomic analyses by comparing ranges of precision, accuracy, and recall. We show that proper experimental design and analysis parameters can reduce false positives, provide greater resolution of species in complex metagenomic samples, and improve the interpretation of results.

Isolation of total RNA from transgenic mouse melanoma subsets using fluorescence-activated cell sorting.

The majority of tumors, including melanoma, are phenotypically heterogeneous in that they contain various cell populations with differential expression of cell surface antigens such as CD133/Prominin-1. We have used fluorescence-activated cell sorting (FACS) technology to purify CD133(+) and CD133(-) cellular subsets from mouse melanoma models for high-quality total RNA practical for downstream applications such as expression profiling. Implementation of this strategy can lead to higher resolution of transcripts that are potentially important for the survival and functionality of one cancer cell population relative to another. Suboptimal extraction of RNA after FACS is common and can ultimately result in misinterpretations that impede the effective design of novel therapies. Here, we describe a number of methods that have been amenable to the successful isolation of high-quality total RNA after FACS of CD133(+) and CD133(-) mouse melanoma cell fractions.

International Standards for Genomes, Transcriptomes, and Metagenomes

Challenges and biases in preparing, characterizing, and sequencing DNA and RNA can have significant impacts on research in genomics across all kingdoms of life, including experiments in single-cells, RNA profiling, and metagenomics (across multiple genomes). Technical artifacts and contamination can arise at each point of sample manipulation, extraction, sequencing, and analysis. Thus, the measurement and benchmarking of these potential sources of error are of paramount importance as next-generation sequencing (NGS) projects become more global and ubiquitous. Fortunately, a variety of methods, standards, and technologies have recently emerged that improve measurements in genomics and sequencing, from the initial input material to the computational pipelines that process and annotate the data. Here we review current standards and their applications in genomics, including whole genomes, transcriptomes, mixed genomic samples (metagenomes), and the modified bases within each (epigenomes and epitranscriptomes). These standards, tools, and metrics are critical for quantifying the accuracy of NGS methods, which will be essential for robust approaches in clinical genomics and precision medicine.

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.