Quality control recommendations for RNASeq using FFPE samples based on pre-sequencing lab metrics and post-sequencing bioinformatics metrics.

BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) tissues have many advantages for identification of risk biomarkers, including wide availability and potential for extended follow-up endpoints. However, RNA derived from archival FFPE samples has limited quality. Here we identified parameters that determine which FFPE samples have the potential for successful RNA extraction, library preparation, and generation of usable RNAseq data. METHODS: We optimized library preparation protocols designed for use with FFPE samples using seven FFPE and Fresh Frozen replicate pairs, and tested optimized protocols using a study set of 130 FFPE biopsies from women with benign breast disease. Metrics from RNA extraction and preparation procedures were collected and compared with bioinformatics sequencing summary statistics. Finally, a decision tree model was built to learn the relationship between pre-sequencing lab metrics and qc pass/fail status as determined by bioinformatics metrics. RESULTS: Samples that failed bioinformatics qc tended to have low median sample-wise correlation within the cohort (Spearman correlation < 0.75), low number of reads mapped to gene regions (< 25 million), or low number of detectable genes (11,400 # of detected genes with TPM > 4). The median RNA concentration and pre-capture library Qubit values for qc failed samples were 18.9 ng/ul and 2.08 ng/ul respectively, which were significantly lower than those of qc pass samples (40.8 ng/ul and 5.82 ng/ul). We built a decision tree model based on input RNA concentration, input library qubit values, and achieved an F score of 0.848 in predicting QC status (pass/fail) of FFPE samples. CONCLUSIONS: We provide a bioinformatics quality control recommendation for FFPE samples from breast tissue by evaluating bioinformatic and sample metrics. Our results suggest a minimum concentration of 25 ng/ul FFPE-extracted RNA for library preparation and 1.7 ng/ul pre-capture library output to achieve adequate RNA-seq data for downstream bioinformatics analysis.

Influence of mosquito genotype on transcriptional response to dengue virus infection.

The mosquito Aedes aegypti is the principal vector that transmits dengue virus (DENV) to humans. The primary factors that trigger a susceptible or refractory interaction of A. aegypti with DENV are not well understood. In this study, our aim is to characterize the influence of vector genotype on differential gene expression of susceptible vs. refractory A. aegypti strains to DENV infection. To accomplish that, we identified differential expression of a set of complementary DNAs (cDNAs; n = 9,504) of the D2S3 (susceptible) and Moyo-D (refractory) strains of A. aegypti to DENV serotype 2 (JAM1409) and compared these results to the differential expression of cDNAs in a different susceptible vector genotype (Moyo-S) relative to the same refractory genotype (Moyo-D) identified from our previous study. We observed that, although the number of differentially expressed transcripts (DETs) was similar in both the studies, about ~95% of the DETs were distinct between Moyo-D/D2S3 vs. Moyo-D/Moyo-S. This suggested that A. aegypti response, to infection of a given genotype of dengue, is largely dependent upon the vector genotype. However, we observed a set of common DETs among the vector strains that were associated with predicted functions such as endocytosis, regulation of autophagy, peroxisome, and lipid metabolism that may be relatively universal in conferring mosquito response to DENV infection.

Stage-specific transcription during development of Aedes aegypti.

BACKGROUND: Aedes aegypti is the most important global vector of dengue virus infection in humans. Availability of the draft genome sequence of this mosquito provides unique opportunities to study different aspects of its biology, including identification of genes and pathways relevant to the developmental processes associated with transition across individual life stages. However, detailed knowledge of gene expression patterns pertaining to developmental stages of A. aegypti is largely lacking. RESULTS: We performed custom cDNA microarray analyses to examine the expression patterns among six developmental stages: early larvae, late larvae, early pupae, late pupae, and adult male and female mosquitoes. Results revealed 1,551 differentially expressed transcripts (DETs) showing significant differences in levels of expression between these life stages. The data suggests that most of the differential expression occurs in a stage specific manner in A. aegypti. Based on hierarchical clustering of expression levels, correlated expression patterns of DETs were also observed among developmental stages. Weighted gene correlation network analysis revealed modular patterns of expression among the DETs. We observed that hydrolase activity, membrane, integral to membrane, DNA binding, translation, ribosome, nucleoside-triphosphatase activity, structural constituent of ribosome, ribonucleoprotein complex and receptor activity were among the top ten ranked GO (Gene Ontology) terms associated with DETs. Significant associations of DETs were also observed with specific KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway modules. Finally, comparisons with the previously reported developmental transcriptome of the malaria vector, Anopheles gambiae, indicated that gene expression patterns during developmental processes reflect both species-specific as well as common components of the two mosquito species. CONCLUSIONS: Our study shows that genes involved in the developmental life cycle of A. aegypti are expressed in a highly stage-specific manner. This suggests that transcriptional events associated with transition through larval, pupal and adult stages are largely discrete.

Comparative expression profiles of midgut genes in dengue virus refractory and susceptible Aedes aegypti across critical period for virus infection.

BACKGROUND: Aedes aegypti is the primary mosquito vector for dengue virus (DENV) worldwide. Infectivity of dengue virus varies among natural populations of this mosquito. How A. aegypti responds to DENV infection relative to which genes and associated pathways contribute to its differential susceptibility as a vector is not well defined. METHODS/PRINCIPAL FINDINGS: Here, we used custom cDNA microarrays to identify groups of genes that were differentially expressed in midgut tissues between susceptible and refractory strains in a highly time specific manner. While genes involved in protein processing in the endoplasmic reticulum, mRNA surveillance, and the proteasome were significantly up-regulated in the susceptible strain, several metabolic processes including glycolysis, glycan biosynthesis and Wnt pathway were active in the refractory strain. In addition, several key signaling genes were expressed as common responsive genes in both susceptible and refractory mosquitoes that may be necessary for signal transduction to trigger the appropriate host response to the viral infection. These are coordinately expressed in the form of tight gene networks and expression clusters that may be necessary to differentially contribute to the progression of dengue infection between the two strains. CONCLUSIONS: Our data show that highly correlated differential expression of responsive genes throughout the post infection period in A. aegypti midgut tissues is necessary for a coordinated transcriptional response of the mosquito genes to host or defend the viral infection.

Global cross-talk of genes of the mosquito Aedes aegypti in response to dengue virus infection.

BACKGROUND: The mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in humans, and DENV is the most important arbovirus across most of the subtropics and tropics worldwide. The early time periods after infection with DENV define critical cellular processes that determine ultimate success or failure of the virus to establish infection in the mosquito. METHODS AND RESULTS: To identify genes involved in these processes, we performed genome-wide transcriptome profiling between susceptible and refractory A. aegypti strains at two critical early periods after challenging them with DENV. Genes that responded coordinately to DENV infection in the susceptible strain were largely clustered in one specific expression module, whereas in the refractory strain they were distributed in four distinct modules. The susceptible response module in the global transcriptional network showed significant biased representation with genes related to energy metabolism and DNA replication, whereas the refractory response modules showed biased representation across different metabolism pathway genes including cytochrome P450 and DDT [1,1,1-Trichloro-2,2-bis(4-chlorophenyl) ethane] degradation genes, and genes associated with cell growth and death. A common core set of coordinately expressed genes was observed in both the susceptible and refractory mosquitoes and included genes related to the Wnt (Wnt: wingless [wg] and integration 1 [int1] pathway), MAPK (Mitogen-activated protein kinase), mTOR (mammalian target of rapamycin) and JAK-STAT (Janus Kinase - Signal Transducer and Activator of Transcription) pathways. CONCLUSIONS: Our data revealed extensive transcriptional networks of mosquito genes that are expressed in modular manners in response to DENV infection, and indicated that successfully defending against viral infection requires more elaborate gene networks than hosting the virus. These likely play important roles in the global-cross talk among the mosquito host factors during the critical early DENV infection periods that trigger the appropriate host action in susceptible vs. refractory mosquitoes.

Genome-based polymorphic microsatellite development and validation in the mosquito Aedes aegypti and application to population genetics in Haiti.

BACKGROUND: Microsatellite markers have proven useful in genetic studies in many organisms, yet microsatellite-based studies of the dengue and yellow fever vector mosquito Aedes aegypti have been limited by the number of assayable and polymorphic loci available, despite multiple independent efforts to identify them. Here we present strategies for efficient identification and development of useful microsatellites with broad coverage across the Aedes aegypti genome, development of multiplex-ready PCR groups of microsatellite loci, and validation of their utility for population analysis with field collections from Haiti. RESULTS: From 79 putative microsatellite loci representing 31 motifs identified in 42 whole genome sequence supercontig assemblies in the Aedes aegypti genome, 33 microsatellites providing genome-wide coverage amplified as single copy sequences in four lab strains, with a range of 2-6 alleles per locus. The tri-nucleotide motifs represented the majority (51%) of the polymorphic single copy loci, and none of these was located within a putative open reading frame. Seven groups of 4-5 microsatellite loci each were developed for multiplex-ready PCR. Four multiplex-ready groups were used to investigate population genetics of Aedes aegypti populations sampled in Haiti. Of the 23 loci represented in these groups, 20 were polymorphic with a range of 3-24 alleles per locus (mean = 8.75). Allelic polymorphic information content varied from 0.171 to 0.867 (mean = 0.545). Most loci met Hardy-Weinberg expectations across populations and pairwise FST comparisons identified significant genetic differentiation between some populations. No evidence for genetic isolation by distance was observed. CONCLUSION: Despite limited success in previous reports, we demonstrate that the Aedes aegypti genome is well-populated with single copy, polymorphic microsatellite loci that can be uncovered using the strategy developed here for rapid and efficient screening of genome supercontig assemblies. These loci are suitable for genetic and population studies using multiplex-PCR.

Comparative analysis of the global transcriptome of Anopheles funestus from Mali, West Africa.

BACKGROUND: Anopheles funestus is a principal vector of malaria across much of tropical Africa and is considered one of the most efficient of its kind, yet studies of this species have lagged behind those of its broadly sympatric congener, An. gambiae. In aid of future genomic sequencing of An. funestus, we explored the whole body transcriptome, derived from mixed stage progeny of wild-caught females from Mali, West Africa. PRINCIPAL FINDINGS: Here we report the functional annotation and comparative genomics of 2,005 expressed sequence tags (ESTs) from An. funestus, which were assembled with a previous EST set from adult female salivary glands from the same mosquito. The assembled ESTs provided for a nonredundant catalog of 1,035 transcripts excluding mitochondrial sequences. CONCLUSIONS/SIGNIFICANCE: Comparison of the An. funestus and An. gambiae transcriptomes using computational and macroarray approaches revealed a high degree of sequence identity despite an estimated 20-80 MY divergence time between lineages. A phylogenetically broader comparative genomic analysis indicated that the most rapidly evolving proteins--those involved in immunity, hematophagy, formation of extracellular structures, and hypothetical conserved proteins--are those that probably play important roles in how mosquitoes adapt to their nutritional and external environments, and therefore could be of greatest interest in disease control.

Channel glass-based detection of human short insertion/deletion polymorphisms by tandem hybridization.

The development and critical evaluation of new technologies for identifying genetic polymorphisms will rapidly accelerate the discovery and diagnosis of disease-related genes. We report a novel way for distinguishing a new class of human DNA polymorphisms, short insertion/deletion polymorphisms (indels). A sensor with cylindrical pores named channel glass in combination with tandem hybridization, which uses a 5'-fluorescent labeled stacking probe and microarray-based short allele-specific oligonucleotide (capture probe) was investigated. This methodology allows indels to be detected individually and rapidly with small quantities of target DNA. This establishes a reliable quantitative test. Approaches for simultaneously hybridizing different targets to arrayed probes, designed to detect various indels in parallel, were examined. Five markers were consistently detected in a single hybridization. Possible factors impeding the hybridization reaction process are discussed.

A comparison of hybridization efficiency between flat glass and channel glass solid supports.

Two different solid supports, channel glass and flat glass, were compared for their affect on the sensitivity and efficiency of DNA hybridization reactions. Both solid supports were tested using a set of arrayed, synthetic oligonucleotides that are designed to detect short insertion/deletion polymorphisms (SIDPs). A total of 13 different human SIDPs were chosen for analysis. Capture probes, designed for this test set, were covalently immobilized on substrates. Hybridization efficiency was assessed using fluorescently labeled stacking probes which were preannealed to the target and then hybridized to the support-bound oligonucleotide array; the hybridization pattern was detected by fluorescence imaging. It was found that structural features of nucleic acid capture probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implications on the hybridization process. Our results demonstrate that channel glass has a number of practical advantages over flat glass including higher sensitivity and a faster hybridization rate.

Variability of DNA microarray gene expression profiles in cultured rat primary hepatocytes.

DNA microarray is a powerful tool in biomedical research. However, transcriptomic profiling using DNA microarray is subject to many variations including biological variability. To evaluate the different sources of variation in mRNA gene expression profiles, gene expression profiles were monitored using the Affymetrix RatTox U34 arrays in cultured primary hepatocytes derived from six rats over a 26 hour period at 6 time points (0 h, 2h, 5h, 8h, 14 h and 26 h) with two replicate arrays at each time point for each animal. In addition, the impact of sample size on the variability of differentially expressed gene lists and the consistency of biological responses were also investigated. Excellent intra-animal reproducibility was obtained at all time points with 0 out of 370 present probe sets across all time points showing significant difference between the 2 replicate arrays (3-way ANOVA, p

Gene expression patterns associated with blood-feeding in the malaria mosquito Anopheles gambiae.

BACKGROUND: Blood feeding, or hematophagy, is a behavior exhibited by female mosquitoes required both for reproduction and for transmission of pathogens. We determined the expression patterns of 3,068 ESTs, representing ~2,000 unique gene transcripts using cDNA microarrays in adult female Anopheles gambiae at selected times during the first two days following blood ingestion, at 5 and 30 min during a 40 minute blood meal and at 0, 1, 3, 5, 12, 16, 24 and 48 hours after completion of the blood meal and compared their expression to transcript levels in mosquitoes with access only to a sugar solution. RESULTS: In blood-fed mosquitoes, 413 unique transcripts, approximately 25% of the total, were expressed at least two-fold above or below their levels in the sugar-fed mosquitoes, at one or more time points. These differentially expressed gene products were clustered using k-means clustering into Early Genes, Middle Genes, and Late Genes, containing 144, 130, and 139 unique transcripts, respectively. Several genes from each group were analyzed by quantitative real-time PCR in order to validate the microarray results. CONCLUSION: The expression patterns and annotation of the genes in these three groups (Early, Middle, and Late genes) are discussed in the context of female mosquitoes' physiological responses to blood feeding, including blood digestion, peritrophic matrix formation, egg development, and immunity.