False negative rates in Drosophila cell-based RNAi screens: a case study

<p>Abstract</p> <p>Background</p> <p>High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention.</p> <p>Results</p> <p>We performed a meta-analysis of several genome-wide, cell-based <it>Drosophila </it>RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene.</p> <p>Conclusions</p> <p>RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.</p

Comparative analysis of the transcriptome across distant species

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters

Generation and characterization of interferon-lambda 1-resistant H1N1 influenza A viruses

<div><p>Influenza A viruses pose a constant potential threat to human health. In view of the innate antiviral activity of interferons (IFNs) and their potential use as anti-influenza agents, it is important to know whether viral resistance to these antiviral proteins can arise. To examine the likelihood of emergence of IFN-λ1-resistant H1N1 variants, we serially passaged the A/California/04/09 (H1N1) strain in a human lung epithelial cell line (Calu-3) in the presence of increasing concentrations of recombinant IFN-λ1 protein. To monitor changes associated with adaptation of this virus to growth in Calu-3 cells, we also passaged the wild-type virus in the absence of IFN-λ1. Under IFN-λ1 selective pressure, the parental virus developed two neuraminidase (NA) mutations, S79L and K331N, which significantly reduced NA enzyme activity (↓1.4-fold) and sensitivity to IFN-λ1 (↓˃20-fold), respectively. These changes were not associated with a reduction in viral replication levels. Mutants carrying either K331N alone or S79L and K331N together induced weaker phosphorylation of IFN regulatory factor 3 (IRF3), and, as a consequence, much lower expression of the IFN genes (<i>IFNB1</i>, <i>IFNL1</i> and <i>IFNL2/3</i>) and proteins (IFN-λ1 and IFN-λ2/3). The lower levels of IFN expression correlated with weaker induction of tyrosine-phosphorylated STAT1 and reduced RIG-I protein levels. Our findings demonstrate that influenza viruses can develop increased resistance to the antiviral activity of type III interferons.</p></div

Generation and characterization of interferon-lambda 1-resistant H1N1 influenza A viruses - Fig 6

<p><b>(A, B, C) Influenza-induced IFN gene expression levels in Calu-3 cells.</b> Cells were infected with the indicated recombinant viruses (MOI = 1) and the levels of IFNs were quantified by qPCR at 12, 48, and 72 hpi. Values were determined by comparison to standard curves for each gene and the results are expressed as RNA copy numbers. *<i>P</i> < 0.05; °<i>P</i> < 0.01, compared to the values for the CA/04 virus. <b>(D, E) IFN-λ1 and –λ2/3 protein production in Calu-3 cells.</b> Cells were infected with viruses at a MOI of 5. Supernatants were collected at 24, 48, and 72 hpi, and the levels of secreted IFN-λ1 protein were determined by ELISA. °<i>P</i> < 0.01, compared to the values for the CA/04 virus.</p

Growth characteristics of wild-type and two passaged H1N1 influenza viruses.

<p>Growth characteristics of wild-type and two passaged H1N1 influenza viruses.</p

Replication of the A/California/04/09, CA/04^+IFN-λ1 and CA/04^–IFN-λ1 viruses in Calu-3 cells.

<p>The results are expressed as log₁₀PFU/ml from three independent experiments. °<i>P</i> < 0.01, compared to the values for the wild-type virus.</p

Generation of influenza A viruses with decreased sensitivity to IFN-λ1.

<p>A/California/04/09 virus was passaged in Calu-3 cells in the presence (red line) or absence (blue line) of increasing concentrations of IFN-λ1 (green line). *<i>P</i> < 0.05, °<i>P</i> < 0.01, compared to the values for the CA/04^–IFN-λ1 virus.</p

Nucleotide and amino acid substitutions identified in selected H1N1 influenza viruses.

<p>Nucleotide and amino acid substitutions identified in selected H1N1 influenza viruses.</p

Growth characteristics and NA enzymatic properties of recombinant H1N1 influenza viruses.

<p>Growth characteristics and NA enzymatic properties of recombinant H1N1 influenza viruses.</p

Generation and characterization of interferon-lambda 1-resistant H1N1 influenza A viruses - Fig 4

<p><b>(A) Receptor specificity of A/California/04/09, CA/04</b>^{<b>+IFN-λ1</b>}, <b>and CA/04</b>^{<b>–IFN-λ1</b>}<b>viruses.</b> *<i>P</i> < 0.05, compared to the values for the wild-type virus. <b>(B) Polymerase activity of RNP complexes of wild-type and CA/04</b>^{<b>–IFN-λ1</b>} <b>containing PA V14I mutation.</b> The values represent the means ± standard deviations of activity of each RNP complex relative to that of CA/04 virus. °<i>P</i> < 0.01, compared to the values for the CA/04 virus. <b>(C) Replication efficiency of CA/04, CA/04-M1</b>^{<b><i>A183G</i></b>}, <b>and CA/04-M2</b>^<b>E70K</b> <b>viruses in Calu-3 cells.</b> Representative results expressed as log₁₀PFU/ml from three independent experiments are shown. *<i>P</i> < 0.05, compared to the values for the CA/04 virus. <b>(D) Antiviral activity of IFN-λ1 against CA/04, CA/04-M1</b>^{<b><i>A183G</i></b>}, <b>and CA/04-M2</b>^<b>E70K</b> <b>viruses as measured by cell ELISA.</b></p