PlantExpress

ArthaExperss OryzaExpress

User’s guide

Ver.2_161025

keyword Search

1. Content

BLAST

MyList

Gene Expression Network

Advanced Search

Publications

ID Converter

Help

Download

Section 4.

Section 2.

Section 3.

Section 5.

Publication List about Express DB series

Section 6.

Help

Bulk download

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PlantExpress (Cross-species)PlantExpress provides functions to compare Gene Expression Networks (GENs) constructed with

OryzaExpress and ArthaExpress on the basis of ortholog relationship.

ArthaExpress (Single-species)Sub-platform of PlantExpress for a GEN analysis in

Arabidopsis thaliana.

OryzaExpress (Single-species)Sub-platform of PlantExpress for a GEN analysis in

Oryza sativa (rice).

These platforms can be selected in the header section.

2-1. How to Keyword SearchKeyword search can be performed in Top page or Advanced search. Keyword search of Top page (Quick Search) can selected search target for Transcript or Knowledge-Based Functional Description (KBFD) or Biological Condition of Microarray Sample (Microarray sample). In Advanced search, it is possible to perform complicated search that combines Boolean search ( ”AND”, “OR”, “NOT” ) and Search Field in additional to the Keyword. Useful as keywords, ID (Locus, Probe), the life event of the plant (flowering, germination, ..), plant anatomy (leaf, root, shoot, ..), environmental stress, compounds, and so on.

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① Keyword Search of PlantExpress

Select Search target, input Keywords, please click .This search is performed against ArthaExpress and OryzaExpress, Search Result link to each database.

Advanced Search

2. keyword Search (Gene, KBFD, Microarray sample)

PlantExpress

SEARCH

EXAMPLE

② Quick Search of ArthaExpress and OryzaExpress Top Page

Select the target that you want to search, enter Keywords.If you have no any keyword, please try click .

③ Advanced Search of ArthaExpress and OryzaExpress

Select search field, enter Keywords. Add keyword query boxDelete keyword query box

ArthaExpress OryzaExpress

2-2. Gene Search Result

①In addition, if you want to search the results, enter

keywords, select the AND or OR, please click .

② Shown fields

Field of display / non-display can be selected.

③ Download / Add to list

It is possible to add the search results to MyList or download tsv file of search results.Please see also MyList.

④ Result Description

To the field, you can filter by using keywords.You can add the selected Probe ID to MyList.

Gene search is performed against the annotations of Genes in ArthaExpress or OryzaExpress ( LocusID, Probe ID / Knowledge-Based Functional Description / Functional Description / Related IDs, Names / GO / KEGG Orthology / InterPro ), and the results of that search are combined with the original query if you created in advanced search. Shown screenshot is the Gene Search Result of a search using the keyword “leaf”. Words that match the query are highlighted in yellow. When you click the Locus ID or Probe ID, and then move to the Locus information page (section 2-3-1), Probe information page (section 2-3-2) .

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• Locus ID• Functional Description• Related ID / Names • Knowledge-Based Functional Description : Please see Knowledge-Based Functional Description Search Result (section 2-4).• Expression Profile : Please see Expression Profile (section 2-3-3).• Gene Ontology• KEGG Pathway• InterPro

2-3. Information detail2-3-1. Locus information detail

2-3-2. Probe information detail• Probe ID• Corresponding Gene Locus• Other Probes for Gene Locus : When the corresponding multiple of probes to one of the same locus, can be compared the expression

pattern between probes. With regard to the operation of Expression profile graph, please see Expression Profile (section 2-3-3).• Neighborhoods of Probe on GEN : XX Probe(s) : Expression similarity was determined by GEN’s three calculation methods ( Pearson

correlation coefficient (PCC), Partial correlation coefficient (PAC), Distance in CA space (DCA) ). Number of graph title(XX) indicates total number of expression similarity probes. Probe lists shows expression similar probes derived at each calculation. When you click the list of check boxes, you can compare the expression patterns on the expression profile graph. With regard to the operation of Expression profile graph, please see Expression Profile (section 2-3-3).

2-3-3. Expression Profile

• CONFIG•X-axis Sorting : Experiment type / Tissue / Stage /

Genetic background / Genetic Modification / Series acc. •Background Color : ON / OFF•Y-axis Scaling : None (default) / log10 / max

None (default) : The expression levels are represented in FPKM without scaling.

log10 : The expression levels are represented in logarithm of FPKM values to base 10. Before the logarithm calculation, 1 is added to all FPKM values to avoid invalid calculation.

/max : The expression level of a transcript are represented in a relative expression level by dividing with the maximum FPKM value of the transcript among the all Runs.

•Download•SAMPLE information >> Please see also section 2-5

• Sample Acc.• Series Acc.• Platform• Experiment type• Tissue• Stage• Genetic background• Genetic Modification• Genetic Modification Type• Treatment detail

•EXP. VALUE•Value of Signal

Shoot

Expression Profile has calculated expression level of transcripts by using Microarray (ArthaExpress Platform : Affymetrix, OryzaExpressPlatform : Agilent Rice Gene Expression 4x44K Microarray and Affymetrix Rice Genome Array), expression level of each Samples shown in the graph. X-axis shows Microarray Sample ID, Y-axis shows Expression levels. Sample description (Experimental details) is manually curated from GEO and literature (Please see also section 2-5). Plot area coloring shows category of X-axis sorting key ( Experiment type / Tissue / Stage / Genetic background / Genetic Modification / Series acc. ). For example, thin pink of left side area of Screenshot indicate microarray sample is used tissue of shoot. In addition, bottom small graph select given area, you can expand the expression profile graph in the x-axis direction.

2-4. Knowledge-Based Functional Description Search ResultKnowledge-Based Functional Description (KBFD) search is focus on the KBFD and only views KBFD results. This search is performed against the KBFD information ( Entry term / Relation term / Keyword for searching paper ). Shown screenshot is the KBFD Search Result of a search using the keyword “drought”.

①In addition, if you want to search the results, enter

keywords, select the AND or OR, please click .

② Shown fields

Field of display / non-display can be selected.

③ Result Description

To the field, you can filter by using keywords.

④ Relationship (Entry1 – Relation – Entry2)

⑤ Source sentence

Relationships was extracted from the sentence.

⑥ Keyword for Article

Keyword that was used to query search the literature in PubMed. Query e.g., “Species” AND “Keyword”.

⑦ Source paper

Link to PubMed literature cited the sentence.

⑧ Corresponded locus

This information indicates corresponding Gene Symbol to Locus ID based on Evidence.

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2-5. Biological Condition of Microarray Sample Search Result

Manually curated microarray experimental description• Sample ID / Series ID / Platform : link to GEO• Experiment type : Classification of sample series that what kind of experiment type. Classification based on the Plant Environmental ontology

(EO) and distinctive standard.• Tissue : Plant tissue (Classification based on the Plant ontology (PO)).• Developmental stage : Plant sampling or treatment stage.• Genetic background : Cultivar, Combination hybrid, line number, etc.• Genetic Modification detail : Transformed or mutation genes, vector construct, Genotype, Phenotype, etc.• Genetic Modification Type : Mutation type, type of inbred, etc.• Treatment detail : Treatment detail

Microarray sample search is performed against microarray experimental details ( Sample ID, Series ID / Platform / Experiment type / Tissue / Developmental stage / Genetic detail / Treatment detail ) obtained by manual curation from GEO and Literature. Words that match the query are highlighted in yellow. When you click the Sample ID or Series ID or Platform ID, and then link to the GEO information page.

①In addition, if you want to search the results, enter

keywords, select the AND or OR, please click .

② Shown fields

Field of display / non-display can be selected.

③ Download / Add to list

It is possible to add the search results to MyList (RUN List) or download tsv file of search results.Please see also MyList.

④ Result Description

To the field, you can filter by using keywords.You can add the selected Sample ID to MyList.

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3-1. How to BLAST Search

1. Input query sequence (nucleotide or amino acid) or upload sequence file, select TEXT or UPLOAD2. Select Options

BLAST Program : blastn / blastp / blastx / tblastn / tblastxFilter E-Value MAX number of hits / query

3. Click SUBMIT

3. BLAST

3-2. BLAST Search Result

Click bit score bar, jump to Align View.

Mouse over bit score bar, pop-up BLAST score (identity, E-value, Bit score).

BLAST Search Result shows “Graphical View”, “Align View” and “List View”. Transcript ID click link to corresponded Locus information (section 2-3-1).

4-1. How to use MyListMyList can make two kinds of list, Probe list and Microarray Sample List. By making your Lists, it is possible to perform various genetic analysis in PlantExpress, ArthaExpress, OryzaExpress against list, you can easily download the results, sequence data, functional annotation information.

4-1-1. New list make from MyList for

4-1-2. New list make from Search Result and add Probe IDs or Sample IDs to existing list

1. Click New List.2. Input List name, click .3. Click to submit Locus IDs / Transcript IDs / Gene names by text. If your query included the Gene Name or Locus

ID, correspondence between the transcript is required. Next step, you must execute select the transcript.4. Select Transcript ID to add to the List.

4-1-3. List ConfigurationList name click, open the Transcript list and configuration icons.

1. Click or on Search Result Page, pop-up List select field.2. To make new list, input List name, click New, add to list Probe IDs or Sample IDs.3. To add to the existing list, select target list, add to list Probe IDs or Sample IDs.

ADD TO LIST

Transcript List : Same list format as Gene Search Results (section 2-2). Download transcript list : xls file, tsv file, cDNA fasta file, Protein fasta fileUpload transcript list : Please see section 4-1-1.Rename this listSend to GEN viewer : Please see GEN (section 5)Delete this list

4. MyList

SUBMIT

ArthaExpress OryzaExpress

4-2. My List ToolsOryza4-2-1. Gene Set Interpretation

•Enrichment TestEnrichment Test can examine an enrichment of gene functions such as GO terms and KEGG pathways in a probe list. In order to perform the Enrichment Test, you must select the probe list, microarray platforms (OryzaExpress only) and test target (GO or KEGG pathways). • GO ID• GO Term• Type : Biological process, Cellular component, Molecular Function• Num. of gene : Number of genes corresponding to the probes list and assigned with the GO term.• Background : Background refers to the number of all probes of the selected platform has been assigned to the GO term.• p-value : p-value of Fisher’s exact test, you can select three types of threshold (none, 1%, 5%). • Q-value : Q-value obtained by the Q-value method, you can select three types of threshold (none, 1%, 5%).

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4-2-2. Gene Expression Network Analysis•Network ConstructionNetwork Construction can construct GENs using a user-selected dataset combination (Probe and Sample).1. Select Calculation Method (PCC or DCA). You can not select PAC in this tool. Then, you can select analysis target all probes or only probe list.2. Select Probe list3. Select Sample list4. Select Platform (OryzaExpress only)• GEN viewer : Please see section 5-3• NODES : Probe(Node) information• ACTION : Regulation of threshold, Node layout change, Export for Network Analysis in json format file• EXPRESSION PROFILE :When you select node(s), Expression Profile graph is displayed. (Section 2-3-3)

Artha Oryza

Calculated by DCA, you can show Histogram of distance in CA space. You can change threshold watching this graph. Threshold of percentile change to drag the green circle in horizontal direction.

Actual threshold

•Network Analysis (OryzaExpress, ArthaExpress and PlantExpress selected GENs each species) This function is used to isolate each independent GEN from complexed GEN information (see also figure), which is constructed with a user-selected dataset in the ‘Network Construction’ or pre-calculated using all data. The results of GEN that have been created in this tool is output as probe lists. Calculation method and threshold is same to the GEN viewer (section 5-2).

4-2-2. Gene Expression Network Analysis Artha Oryza

Network Analysis

Artha Oryza4-2-3. Basic Gene Expression Analysis

•Gene Expression ProfileGene Expression Profile’ is used to browse expression profile of selected probe list in selected microarray samples as a line graph and to download the expression data as an image file or a tsv file. Please see also section 2-3-3.

•MA PlotThe ‘MA plot’ is used to visualize intensity ratio (M) and average intensity (A) between two selected microarray samples in a scatter plot, where ‘M’ and ‘A’ are represented in the horizontal and vertical axes, respectively. You can select Probe list, Sample List, Platform, two microarray samples.

Navi : Change the scale of plot area.

Filtering : Filter by signal intensity and log2 ratio.

Drag and select plot

Artha Oryza4-2-3. Basic Gene Expression Analysis

• t-TestThe ‘t-Test’ is used to explore probes that have significantly different expression levels between two sample lists by Welch’s t-test followed by controlling the false discovery rate.• Probe• Sample value• p-value : Calculate p-value by Welch Two Sample t-test, you can select three types of threshold (none, 1%, 5%). • FDR : Calculate FDR by Benjamini & Hochberg method, you can select three types of threshold (none, 1%, 5%). • Boxplot : Click , shown Boxplot graph about the selected probe.

•Correspondence AnalysisThe ‘Correspondence Analysis’ is used to classify probes and samples based on the expression profiles. In a resulting space (plotting area), the more similar profiles the probes or microarray samples have, the closer coordinate the probes or samples are plotted at.The red plot means the Probe. Information of the selected probe is shown at the bottom of the table (PROBE tab and EXPRESSION PROFILE tab). The blue plot means the microarray sample. Information of the selected sample is shown at SAMPLE tab.

BOXPLOT

Drag and select plot

4-2-4. List Management• List OperationsThe ‘List Operation’ is used to obtain union, intersection, and subtraction of probe or sample lists.

•Export / Import / DeleteThe ‘Export/Import/Delete’ is mainly used to export all probe lists and sample lists in bulk as a local file for backup, and import the file.

4-2-5. PlantExpress MyList tools•Ortholog / ParalogOrtholog/Paralog is used to obtain probes for orthologous or paralogous genes.

•Network AnalysisWhereas the submission form of this analytical function is almost same with that of the ‘Network Analysis’ of the single-species mode (section 4-2-2), an extra option ‘GENs conserved species’ is available in cross-species mode (see also figure).

•Network ViewerGEN Viewer -> Action -> Upload a network fileUpload a network file exported from ‘Network construction’function (filename extension is .compress.json file ) or downloadFunction on this viewer.

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Gene Expression Network (GEN) can be viewed graphical network between Probes by using GEN viewer. Type of network represented by GEN is Ortholog / Paralog relationship ( Arabidopsis thaliana vs Oryza sativa, this function use only PlantExpress ) and expression similarity (calculated by PCC, PAC, DCA). You can make GEN by two methods ( GEN page (in this section) or MyList ). GEN that you create in this section is targeted at all microarray samples. If you will select any samples and created GEN, please use MyList -> Tools -> Network Construction (section 4-2-2).

5. Gene Expression Network (GEN)5-1. How to make GEN

GEN of ArthaExpress and OryzaExpress is only viewed expression similarity between microarray probes.1. Input Probe ID / Gene namesEnter query / Select probe List / Upload probe list text file2. Select Probe IDIf your query is all Probe ID, this step will be skipped.3. Select Search OptionsGEN viewer / Download Cytoscape format

5-1-1. Gene Expression Network for

5-1-3. MyList1. Select the list you want to view the GEN, Click Send to GEN viewer.2. Select Search Options.GEN viewer / Download Cytoscape format

ArthaExpress OryzaExpress

5-1-2. Gene Expression Network for PlantExpress

GEN of PlantExpress is integrated probe list of ArthaExpress and OryzaExpress.1. Select Source Probe List2. Select OptionsAdd Ortholog Nodes

ArthaExpress OryzaExpress

5-2. Calculating method of GEN (Default value of threshold)

PCC : Greater than 0.7 were extracted as strongly positively correlated probes.PAC : Probe pairs showing Z-score larger than 3.10 were extracted as significantly positively associated probes.DCA : Top 0.1% of probe pairs located most closely together in the space summarized by CA were extracted as probes showing significantly similar profiles.If you will change and determine threshold of GEN, please use MyList -> Tools -> Network Construction (section 4-2-2). This content and MyList-> Tools -> Network Analysis can not change GEN’s threshold (default value setting).

ThresholdArabidopsisAffymetrix

RiceAffymetrix

RiceAgilent

PCC >0.7

PAC > 0.111 > 0.075 > 0.124

DCA < 0.076 < 0.058 < 0.045

In order to construct global GENs employing all available microarray data, the Euclidian distance in correspondence analysis space (DCA), Pearson correlation coefficient (PCC), and partial correlation coefficient (PAC) were calculated.

5-3. GEN Viewer

5-3-1. GEN Viewer Legend (default setting)

Node color Blue : Arabidopsis thaliana gene (from ArthaExpress)Node color Green : Oryza sativa gene (from OryzaExpress)

In the GEN viewer, a gene is shown as a node (a circle), and a relationship of Ortholog / Paralog or similar expression profiles between two genes is shown as an edge (a line) connection two nodes. Nodes and edges color, kind of edge, network layout, and so on can be changed from the default setting.

5-3-2. GEN Viewer Config

Edge color Blue : Similar expression profile (from ArthaExpress)Edge color Green : Similar expression profile (from OryzaExpress)Dotted edge (Gray) : Relationship of Ortholog (Shown only PlantExpress)Dotted edge (same color to the node) : Relationship of Paralog (Shown only PlantExpress)

• View Config (nodes)• Node color• Node size• Node Visibility : Select the Nodes having at least one edge, can not show nodes not have any edge. Move to the all, all nodes show the

GEN viewer. • Label type : Display or non-display a node label (Select Locus ID / Related ID)• Label color• Label size

• View Config (edges)• Evidence Platform : Affymetrix and Agilent / Affimetrix / Agilent• Evidence Statistics : DCA, PCC, PAC / DCA / PCC / PAC• Edge Width• Edge Style : solid / dashed / dotted

• View Config (global)• Background color

Right side of GEN viewer, can be adjusted graphical interface of viewer.

5-3-3. GEN Viewer operation

5-3-4. GEN Viewer operation toolGene Expression : Shown expression profiles of select node (section 2-3-3).Node List : Shown node list Gene Search Result format (section 2-2).Config : Please see section 5-3-2.Action• Upload a network file : • Download : • Save probes corresponded to selected locus : as Mylist• Omit selected nodes :• Relayout network : default / spread / concentric / grid / circle / cose / breadthfirst• History : You can back to the previous work.

GEN data : Statistics information of GEN (Number of nodes, Number of edges) window display / non-display Legend : Network Legend window (section 5-2-1) display / non-display

①Navigation button②Fit-all ③Zoom in / out④Select the node, move the node and drag.⑤Click a node, shown node information.⑥Click an edge, shown edge information.⑦Right-click a node, shown node option.

Locus information : Jump to locus page (section 2-3-1).Expand from this node : GEN expand 1-pass from this node.Omit this node :Select connected nodes :Edit style : Change node style.

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6. ID Converter6-1. How to use ID CoverterID Converter can be converted to a variety of corresponding IDs. ① Locus ID → Microarray probe ID②Microarray probe ID → Locus ID③ Locus ID → another types locus ID

1. Select Query Type• Locus ID

: TAIR ID (e.g., AT2G16800): RAP Locus ID (IRGSP-1.0 e.g., Os04g0556500) or MSU Locus ID (RGAP7 e.g., LOC_Os04g46980.1)

• Probe ID: Affimetrix microarray probe ID (e.g., 265353_at): Affimetrix microarray probe ID (e.g., Os.11501.1.S1_at) or

Agilent microarray probe accession (e.g., Os04g0556500|mRNA|AK120794|CDS+3'UTR)

2. Need information of un-specific probes? (for query of Locus ID)• No, I need only locus-specific probes.• Yes, I’d like to search all probes (both locus-specific and cross-hybridyzable probes).

3. Query ID listEnter query / Select probe List / Upload probe list text file

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