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Characterization of the specific expression and chromatin profiles of genes enables understanding how they contribute to tissue/organ development and the mechanisms leading to diseases. Whilst the number of single-cell sequencing studies is increasing dramatically; however, data mining and reanalysis remains challenging. Herein, we systematically curated the up-to-date and most comprehensive datasets of sequencing data originating from 2760 bulk samples and over 5.1 million single-cells from multiple developmental periods from humans and multiple model organisms. With unified and systematic analysis, we profiled the gene expression and chromatin accessibility among 481 cell-types, 79 tissue-types and 92 timepoints, and pinpointed cells with the co-expression of target genes. We also enabled the detection of gene(s) with a temporal and cell-type specific expression profile that is similar to or distinct from that of a target gene. Additionally, we illustrated the potential upstream and downstream gene−gene regulation interactions, particularly under the same biological process(es) or KEGG pathway(s). Thus, TEDD (Temporal Expression during Development Database), a value-added database with a user-friendly interface, not only enables researchers to identify cell-type/tissue-type specific and temporal gene expression and chromatin profiles but also facilitates the association of genes with undefined biological functions in development and diseases. The database URL is https://TEDD.obg.cuhk.edu.hk/.

Background Birth defects pose a major challenge to infant health. Thus far, however, the causes of most birth defects remain cryptic. Over the past few decades, considerable effort has been expended on disclosing the underlying mechanisms related to birth defects, yielding myriad treatises and data. To meet the increasing requirements for data resources, we developed a freely accessible birth defect multi-omics database (BDdb, http://t21omics.cngb.org) consisting of multi-omics data and potential disease biomarkers. Results In total, omics datasets from 136 Gene Expression Omnibus (GEO) Series records, including 5245 samples, as well as 869 biomarkers of 22 birth defects in six different species, were integrated into the BDdb. The database provides a user-friendly interface for searching, browsing, and downloading data of interest. The BDdb also enables users to explore the correlations among different sequencing methods, such as chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) from different studies, to obtain the information on gene expression patterns from diverse aspects. Conclusion To the best of our knowledge, the BDdb is the first comprehensive database associated with birth defects, which should benefit the diagnosis and prevention of birth defects.

Precision medicine relies on high-accuracy individual-level genotype data. However, the whole-genome sequencing (WGS) is currently not suitable for studies with very large sample sizes due to budget constraints. It is particularly important to construct highly accurate haplotype reference panel for genotype imputation. In this study, we selected 9,950 individuals from the China Kadoorie Biobank (CKB) cohort and 50 Chinese samples from the 1000 Genome Project (1KGP) for medium-depth WGS to construct a CKB reference panel. The results of imputing microarray datasets showed that the CKB panel outperformed the extended high coverage 1KGP, TOPMed, ChinaMAP, and NuyWa panels in terms of both the number of well-imputed variants and imputation accuracy. In addition, we have completed the imputation of over 100,000 CKB microarray data with the CKB panel, and the after-imputed genotype data is the hitherto largest whole genome data of the Chinese population. Finally, we developed an online server for offering free genotype imputation service based on the CKB reference panel (https://db.cngb.org/imputation/). We believe that the constructed CKB reference panel is of great value for imputing microarray or low-depth genotype data of Chinese population. The imputation-completed 100,000 microarray data are fundamental resources of population genetic studies for complex traits and diseases in the Chinese population.

Viral infectious diseases are a devastating and continuing threat to human and animal health. Receptor binding is the key step for viral entry into host cells. Therefore, recognizing viral receptors is fundamental for understanding the potential tissue tropism or host range of these pathogens. The rapid advancement of single-cell RNA sequencing (scRNA-seq) technology has paved the way for studying the expression of viral receptors in different tissues of animal species at single-cell resolution, resulting in huge scRNA-seq datasets. However, effectively integrating or sharing these datasets among the research community is challenging, especially for laboratory scientists. In this study, we manually curated up-to-date datasets generated in animal scRNA-seq studies, analyzed them using a unified processing pipeline, and comprehensively annotated 107 viral receptors in 142 viruses and obtained accurate expression signatures in 2 100 962 cells from 47 animal species. Thus, the VThunter database provides a user-friendly interface for the research community to explore the expression signatures of viral receptors. VThunter offers an informative and convenient resource for scientists to better understand the interactions between viral receptors and animal viruses and to assess viral pathogenesis and transmission in species. Database URL: https://db.cngb.org/VThunter/.

The brain of the domestic pig (Sus scrofa domesticus) has drawn considerable attention due to its high similarities to that of humans. However, the cellular compositions of the pig brain (PB) remain elusive. Here we investigated the single-nucleus transcriptomic profiles of five regions of the PB (frontal lobe, parietal lobe, temporal lobe, occipital lobe, and hypothalamus) and identified 21 cell subpopulations. The cross-species comparison of mouse and pig hypothalamus revealed the shared and specific gene expression patterns at the single-cell resolution. Furthermore, we identified cell types and molecular pathways closely associated with neurological disorders, bridging the gap between gene mutations and pathogenesis. We reported, to our knowledge, the first single-cell atlas of domestic pig cerebral cortex and hypothalamus combined with a comprehensive analysis across species, providing extensive resources for future research regarding neural science, evolutionary developmental biology, and regenerative medicine.

A high-quality genome variation database derived from a large-scale population is one of the most important infrastructures for genomics, clinical and translational medicine research. Here, we developed the Chinese Millionome Database (CMDB), a database that contains 9.04 million single nucleotide variants (SNV) with allele frequency information derived from low-coverage (0.06×–0.1×) whole-genome sequencing (WGS) data of 141 431 unrelated healthy Chinese individuals. These individuals were recruited from 31 out of the 34 administrative divisions in China, covering Han and 36 other ethnic minorities. CMDB, housing the WGS data of a multi-ethnic Chinese population featuring wide geographical distribution, has become the most representative and comprehensive Chinese population genome database to date. Researchers can quickly search for variant, gene or genomic regions to obtain the variant information, including mutation basic information, allele frequency, genic annotation and overview of frequencies in global populations. Furthermore, the CMDB also provides information on the association of the variants with a range of phenotypes, including height, BMI, maternal age and twin pregnancy. Based on these data, researchers can conduct meta-analysis of related phenotypes. CMDB is freely available at https://db.cngb.org/cmdb/.

Breast cancer is one of the most common cancers, accounting for about 30% of female cancers and a mortality rate of 15%. The 5-year survival rate is most commonly used to assess cancer progression and guide clinical practice. We used the CatBoost model to systematically construct a five-year mortality risk prediction model based on two independent data sets (BRCA_METABRIC, BRCA_TCGA). The model input data are the somatic genomic variants (copy number variation, SNP locus, cumulative mutation number of genes) and phenotype data of cancer samples. The optimal model combined all the above characteristics, and the AUC reached 0.70 in an independent external data set. At the same time, we also conducted a biological analysis of the characteristics of the model and found some potential biomarkers (TP53, DNAH11, MAP3K1, PHF20L1, etc.). The results of model risk stratification can be used as a guide for the prognosis of breast cancer.

Flowering transition is pivotal and tightly regulated by complex gene-regulatory-networks, in which AGL16 plays important roles. But the molecular function and binding property of AGL16 is not fully explored in vivo. With ChIP-seq and comparative transcriptomics approaches, we characterized the AGL16 targets spectrum and tested its close molecular and genetic interactions with SOC1, the key flowering integrator. AGL16 bound to promoters of more than 2000 genes via CArG-box motifs that were highly similar to that of SOC1. Being consistent with this, AGL16 formed protein complex and shared a common set of targets with SOC1. However, only very few genes showed differential expression in the agl16-1 loss-of-function mutant, whereas in the soc1-2 knockout background, AGL16 repressed and activated the expression of 375 and 182 genes, respectively, with more than a quarter of the DEGs were also bound by AGL16. AGL16 targeted potentially to about seventy flowering time genes involved in multiple pathways. Corroborating with these, AGL16 repressed the flowering time stronger in soc1-2 than in Col-0 background. These data reveals that AGL16 regulates gene expression and flowering time with a partial dependency on SOC1 activity. Moreover, AGL16 participated in the regulation of water loss and seed dormancy. Our study thus defines the AGL16 molecular spectrum and provides insights underlining the molecular coordination of flowering and environmental adaptation.

Background Finding peptides with high binding affinity to Class I major histocompatibility complex (MHC-I) attracts intensive research, and it serves a crucial part of developing a better vaccine for precision medicine. Traditional methods cost highly for designing such peptides. The advancement of computational approaches reduces the cost of new drug discovery dramatically. Compared with flourishing computational drug discovery area, the immunology area lacks tools focused on in silico design for the peptides with high binding affinity. Attributed to the ever-expanding amount of MHC-peptides binding data, it enables the tremendous influx of deep learning techniques for modeling MHC-peptides binding. To leverage the availability of these data, it is of great significance to find MHC-peptides binding specificities. The binding motifs are one of the key components to decide the MHC-peptides combination, which generally refer to a combination of some certain amino acids at certain sites which highly contribute to the binding affinity. Result In this work, we propose the Motif Activation Mapping (MAM) network for MHC-I and peptides binding to extract motifs from peptides. Then, we substitute amino acid randomly according to the motifs for generating peptides with high affinity. We demonstrated the MAM network could extract motifs which are the features of peptides of highly binding affinities, as well as generate peptides with high-affinities; that is, 0.859 for HLA-A*0201, 0.75 for HLA-A*0206, 0.92 for HLA-B*2702, 0.9 for HLA-A*6802 and 0.839 for Mamu-A1*001:01. Besides, its binding prediction result reaches the state of the art. The experiment also reveals the network is appropriate for most MHC-I with transfer learning. Conclusions We design the MAM network to extract the motifs from MHC-peptides binding through prediction, which are proved to generate the peptides with high binding affinity successfully. The new peptides preserve the motifs but vary in sequences.

The genetically engineered pig is regarded as an optimal source of organ transplantation for humans and an excellent model for human disease research, given its comparable physiology to human beings. A myriad of single-cell RNA sequencing (scRNA-seq) data on humans has been reported, but such data on pigs are scarce. Here, we apply scRNA-seq technology to study the cellular heterogeneity of 3-month-old pig lungs, generating the single-cell atlas of 13,580 cells covering 16 major cell types. Based on these data, we systematically characterize the similarities and differences in the cellular cross-talk and expression patterns of respiratory virus receptors in each cell type of pig lungs compared with human lungs. Furthermore, we analyze pig lung xenotransplantation barriers and reported the cell-type expression patterns of 10 genes associated with pig-to-human immunobiological incompatibility and coagulation dysregulation. We also investigate the conserved transcription factors (TFs) and their candidate target genes and constructed five conserved TF regulatory networks in the main cell types shared by pig and human lungs. Finally, we present a comprehensive and openly accessible online platform, ScdbLung. Our scRNA-seq atlas of the domestic pig lung and ScdbLung database can guide pig lung research and clinical applicability.

The function of nuclear-localized mitochondria remains unknown. We found that mitochondria assembled dense particles to fragment and disperse into the particles, which reassembled to initiate nuclear formation and development. Individually-formed nuclei in one single cell were joined together by mitochondrial fragmentation concurrently partitioning cytoplasm to form an intranuclear inclusion (INC), whose creation was not related to herniation or invagination of the cytoplasm. Along with the nuclear transition of a mitochondrion and its neighboring counterparts, the organelle included itself in the nucleus to become nuclear mitochondrion through peripherally assembling of dense particles. New medium reversed the nuclear formation of the organelles to recovery and re-establishment via the return of the particles, which consisted of dense microvesicles (MIVs).

We previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WDR5 to facilitate capsid nuclear egress. Here, we further show that HCMV infection drives WDR5 to the perinuclear region by a mechanism that requires viral replication and intact microtubules. WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 interacted with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain the increasing WDR5 accumulation in the vAC during infection. WDR5 was then incorporated into HCMV virions and localized to the tegument layer, as demonstrated by fractionation and immune-gold electron microscopy. Thus, WDR5 is driven to the vAC and incorporated into virions, suggesting that WDR5 facilitates HCMV replication at later stage of virion assembly besides the capsid nuclear egress stage. These data highlight that WDR5 is a potential target for antiviral therapy.ImportanceHuman cytomegalovirus (HCMV) has a large (~235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are profoundly reconfigured to establish the virion assembly compartment (vAC), which is important for efficient assembly of progeny virions. We previously reported that WDR5 promotes HCMV nuclear egress. Here, we show that WDR5 is further driven to the vAC and incorporated into virions, perhaps to facilitate efficient virion maturation. This work identified potential roles for WDR5 in HCMV replication in the cytoplasmic stages of virion assembly. Taken together, WDR5 plays a critical role in HCMV capsid nuclear egress and is important for virion assembly, and thus is a potential target for antiviral treatment of HCMV-associated diseases.

Birth defect, not only poses a major challenge for infant health but also attracts the attention of countless people in the world. Chromosome abnormality directly results in diverse birth defects which are generally deleterious and even lethal. Therefore, gaining molecular regulatory insights into these diseases is important and necessary for effective prenatal screening. Recently, with the advance of next-generation sequencing (NGS) techniques, a myriad of treatises and data associated with these diseases are now constantly produced from different laboratories across the world. To meet the increasing requirements for birth-related data resources, we developed a birth defect multi-omics database (BDdb), freely accessible at http://t21omics.cngb.org and consisting of multi-omics data, circulating free DNA (cfDNA) data, as well as diseases biomarkers. Omics data sets from 138 GSE samples, 5271 GSM samples and 328 entries, and more than 2000 biomarkers of 22 birth-defect diseases in 5 different species were integrated into BDdb, which provides a user-friendly interface for searching, browsing and downloading selected data. Additionally, we re-analyzed and normalized the raw data so that users can also customize the analysis using the data generated from different sources or different High-Throughput Sequencing (HTS) methods. To our knowledge, BDdb is the first comprehensive database associated with birth-defect-related diseases. which would benefit the diagnosis and prevention of birth defects.

BackgroundThe specification and differentiation of neocortical projection neurons is a complex process under precise molecular regulation; however, little is known about the similarities and differences in cerebral cortex development between human and mouse at single-cell resolution.ResultsHere, using single-cell RNA-seq (scRNA-seq) data we explore the divergence and conservation of human and mouse cerebral cortex development using 18,446 and 7,610 neocortical cells. Systematic cross-species comparison reveals that the overall transcriptome profile in human cerebral cortex is similar to that in mouse such as cell types and their markers genes. By single-cell trajectories analysis we find human and mouse excitatory neurons have different developmental trajectories of neocortical projection neurons, ligand-receptor interactions and gene expression patterns. Further analysis reveals a refinement of neuron differentiation that occurred in human but not in mouse, suggesting that excitatory neurons in human undergo refined transcriptional states in later development stage. By contrast, for glial cells and inhibitory neurons we detected conserved developmental trajectories in human and mouse.ConclusionsTaken together, our study integrates scRNA-seq data of cerebral cortex development in human and mouse, and uncovers distinct developing models in neocortical projection neurons. The earlier activation of cognition -related genes in human may explain the differences in behavior, learning or memory abilities between the two species.

Background Decapods are an order of crustaceans which includes shrimps, crabs, lobsters and crayfish. They occur worldwide and are of great scientific interest as well as being of ecological and economic importance in fisheries and aquaculture. However, our knowledge of their biology mainly comes from the group which is most closely related to crustaceans – insects. Here we produce a de novo transcriptome database, crustacean annotated transcriptome (CAT) database, spanning multiple tissues and the life stages of seven crustaceans. Description A total of 71 transcriptome assemblies from six decapod species and a stomatopod species, including the coral shrimp Stenopus hispidus, the cherry shrimp Neocaridina davidi, the redclaw crayfish Cherax quadricarinatus, the spiny lobster Panulirus ornatus, the red king crab Paralithodes camtschaticus, the coconut crab Birgus latro, and the zebra mantis shrimp Lysiosquillina maculata, were generated. Differential gene expression analyses within species were generated as a reference and included in a graphical user interface database at http://cat.sls.cuhk.edu.hk/. Users can carry out gene name searches and also access gene sequences based on a sequence query using the BLAST search function. Conclusions The data generated and deposited in this database offers a valuable resource for the further study of these crustaceans, as well as being of use in aquaculture development.

Background Crustacea, the second largest subphylum of Arthropoda, includes species of major ecological and economic importance, such as crabs, lobsters, crayfishes, shrimps, and barnacles. With the rapid development of crustacean aquaculture and biodiversity loss, understanding the gene regulatory mechanisms of growth, reproduction, and development of crustaceans is crucial to both aquaculture development and biodiversity conservation of this group of organisms. In these biological processes, transcription factors (TFs) play a vital role in regulating gene expression. However, crustacean transcription factors are still largely unknown, because the lack of complete genome sequences of most crustacean species hampers the studies on their transcriptional regulation on a system-wide scale. Thus, the current TF databases derived from genome sequences contain TF information for only a few crustacean species and are insufficient to elucidate the transcriptional diversity of such a large animal group. Results Our database CrusTF (http://qinlab.sls.cuhk.edu.hk/CrusTF) provides comprehensive information for evolutionary and functional studies on the crustacean transcriptional regulatory system. CrusTF fills the knowledge gap of transcriptional regulation in crustaceans by exploring publicly available and newly sequenced transcriptomes of 170 crustacean species and identifying 131,941 TFs within 63 TF families. CrusTF features three categories of information: sequence, function, and evolution of crustacean TFs. The database enables searching, browsing and downloading of crustacean TF sequences. CrusTF infers DNA binding motifs of crustacean TFs, thus facilitating the users to predict potential downstream TF targets. The database also presents evolutionary analyses of crustacean TFs, which improve our understanding of the evolution of transcriptional regulatory systems in crustaceans. Conclusions Given the importance of TF information in evolutionary and functional studies on transcriptional regulatory systems of crustaceans, this database will constitute a key resource for the research community of crustacean biology and evolutionary biology. Moreover, CrusTF serves as a model for the construction of TF database derived from transcriptome data. A similar approach could be applied to other groups of organisms, for which transcriptomes are more readily available than genomes. Electronic supplementary material The online version of this article (10.1186/s12864-017-4305-2) contains supplementary material, which is available to authorized users.

Microalgal Chlorella has been demonstrated to process wastewater efficiently from piggery industry, yet optimization through genetic engineering of such a bio-treatment is currently challenging, largely due to the limited data and knowledge in genomics. In this study, we first investigated the differential growth rates among three wastewater-processing Chlorella strains: Chlorella sorokiniana BD09, Chlorella sorokiniana BD08 and Chlorella sp. Dachan, and the previously published Chlorella sorokiniana UTEX 1602, showing us that BD09 maintains the best tolerance in synthetic wastewater. We then performed genome sequencing and analysis, resulting in a high-quality assembly for each genome with scaffold N50 > 2 Mb and genomic completeness ≥91%, as well as genome annotation with 9,668, 10,240, 9,821 high-confidence gene models predicted for BD09, BD08, and Dachan, respectively. Comparative genomics study unravels that metabolic pathways, which are involved in nitrogen and phosphorus assimilation, were enriched in the faster-growing strains. We found that gene structural variation and genomic rearrangement might contribute to differential capabilities in wastewater tolerance among the strains, as indicated by gene copy number variation, domain reshuffling of orthologs involved, as well as a ~1 Mb-length chromosomal inversion we observed in BD08 and Dachan. In addition, we speculated that an associated bacterium, Microbacterium chocolatum, which was identified within Dachan, play a possible role in synergizing nutrient removal. Our three newly sequenced Chlorella genomes provide a fundamental foundation to understand the molecular basis of abiotic stress tolerance in wastewater treatment, which is essential for future genetic engineering and strain improvement.

Eukaryotic nucleic acid binding protein database (ENPD, http://qinlab.sls.cuhk.edu.hk/ENPD/) is a library of nucleic acid binding proteins (NBPs) and their functional information. NBPs such as DNA binding proteins (DBPs), RNA binding proteins (RBPs), and DNA and RNA binding proteins (DRBPs) are involved in every stage of gene regulation through their interactions with DNA and RNA. Due to the importance of NBPs, the database was constructed based on manual curation and a newly developed pipeline utilizing both sequenced transcriptomes and genomes. In total the database has recorded 2.8 million of NBPs and their binding motifs from 662 NBP families and 2423 species, constituting the largest NBP database. ENPD covers evolutionarily important lineages which have never been included in the previous NBP databases, while lineage-specific NBP family expansions were also found. ENPD also focuses on the involvements of DBPs, RBPs and DRBPs in non-coding RNA (ncRNA) mediated gene regulation. The predicted and experimentally validated targets of NBPs have both been recorded and manually curated in ENPD, linking the interactions between ncRNAs, DNA regulatory elements and NBPs in gene regulation. This database provides key resources for the scientific community, laying a solid foundation for future gene regulatory studies from both functional and evolutionary perspectives.

Roses are important plants for human beings with important economical and biological traits like continuous flowering, flower architecture, color and scent, that current model plants do not feature, thus are becoming ideal models for studying these traits. Due to high heterozygosity of rose genomes likely caused by frequent inter-species hybridization, a high-quality and well-annotated genome for Rosa plants is not available yet. Developing genetic and genomic tools with high quality has become necessary for further roses breeding and for disentangling the molecular genetic mechanisms underlying roses domestication. We here generated the high quality and comprehensive reference transcriptomes for Rosa chinensis ‘Old Blush’ (OB) and R. wichuriana ‘Basyes’ Thornless’ (BT), two roses contrasting at several important traits. These reference transcriptomes showed transcripts N50 above 2000bp. The two species shared about 23310 transcripts (N50 = 2364bp), among which about 8975 orthologs were conserved within genera of Rosa. Rosa plants shared about 5049 transcripts (Rosaceae-common) with these from Malus, Prunus, Rubus, and Fragaria. Finally, a pool of 417 transcripts unique to Rosa plants (Rosa-specific) was identified. These Rosaceae-common and Rosa-specific transcripts should facilitate the phylogenetic analysis of Rosaceae plants and investigation of Rosa-specific traits. The data reported here should provide the fundamental genomic tools and knowledge critical for understanding the biology and domestication of roses.

With quantum chemical computation of density functional theory (DFT), the electronic properties including the polarisabilities, polarisability anisotropies and quadrupole moments of a total of 209 congeners of polybrominated diphenyl ethers (PBDEs) were evaluated. The electronic properties were shown to be highly dependent on the bromination pattern, i.e. their values changed sensitively with the number and sites of bromination. Being similar to the 2,3,7,8-, 1,4,6,9-chlorination of dioxins, respectively, 3,3',4,4'-, 2,2',5,5'-bromination of PBDEs can impose relatively greater effects on the electronic properties. Some of electronic properties were found to be potent in explaining the variance of toxicity, and the potency was verified by the development of quantitative structure-activity relationships (QSARs). To further improve the stability and predictability of QSARs for toxicity, two-dimensional topological indices were introduced. In QSARs, polarisability anisotropy was more significant than other polarisability tensors, indicating the implicit occurrence of dispersion interaction between the ligand and aryl hydrocarbon receptor (AhR). For PBDEs, the quadrupole moment was as significant as shown previously for dioxins. As interesting descriptors with encoded information about dispersion and electronics, the electronic properties analysed herein are helpful in obtaining a better understanding of the congener-specific toxicities of PBDEs, and are applicable and may be extended to research into the toxicology of structurally similar compounds, such as halogenated aromatics.

A novel sampling design is proposed that optimizes the spatial configuration of sampling sites and captures maximum intraurban variability in ambient air pollution with a minimum sample size. Unlike the classical sampling design, a deterministic approach is adopted and the redundancy in the site selection is minimized by controlling for spatial autocorrelation. The proposed design was tested and implemented in a medium-sized midwestern city. The analysis suggested that 32 sites were adequate to capture more than 95% of the total variance in airborne particulate 10 microm or less in aerodynamic diameter (PM10). A list of 20 households was prepared around each of the 32 sites. Households were approached in order of their distance from these sites until one was recruited for intensive indoor and outdoor air pollution monitoring from spring through fall of 2008. Finally, 30 households located around the optimal sites participated in the study. One set of four photometric and gravimetric samplers was deployed for each indoor and outdoor environment. The average ambient PM10 concentration (monitored from April to September 2008) at the selected locations was lower but statistically insignificant as compared with the PM10 (computed using the data from mobile sampling in 2006) at the optimal sites.