POTAGE - Popseq Ordered Triticum Aestivum Gene Expression

About POTAGE and the underlying data

Popseq Ordered Triticum Aestivum Gene Expression (POTAGE, pronounced "pəʊˈtɑːʒ") is a web application which integrates map location information with gene expression and inferred functional annotation.

The functionality of POTAGE depends on several datasets:

IWGSC Chromosome Survey Sequences

Consist of individual assemblies of flow-sorted chromosome arms (apart from chromosome 3B assembled as a whole)
Described in A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome, Science 18 July 2014, Vol. 345 no. 6194
Since cited in numerous publications

POPSEQ map of Chromososme Survey Sequences

Utilizes whole genome sequencing for genotyping to establish maker order from sequencing data
Provides the genetic anchoring of the IWGSC survey sequences by population sequencing. For more details, see Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ), Plant J. 76(4) 718-727
Please note that:

Number of contigs per cM interval is dependent on recombination frequency
The resolution of a genetic map is dependent on the population size

High Confidence (HC) wheat gene predictions (a.k.a MIPS gene predictions)

Described in A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome, Science 18 July 2014, Vol. 345 no. 6194
POTAGE utilizes version 2.1

Functional annotation

MIPS annotation provided with the HC predictions
Rice-derived annotation, obtained by

Aligning HC coding sequences to rice proteins from International Rice Genome Sequencing Project (2005), The map-based sequence of the rice genome. Nature 436: 793–800
Annotation retrieved from top rice hits if available and if alignment quality thresholds were met.

RNA-Seq data

The primary RNA-Seq dataset included in the public release of POTAGE is the Chinese Spring tissue series dataset;
Non-oriented library (TruSeq, Illumina) sequenced on Illumina HiSeq2000 2x100bp (PE) for 15 different conditions corresponding to five wheat organs (root, leaf, stem, spike, grain) at three developmental stages each in 2 duplicates


Stage	Wheat growth stage	Feekes scale	Zadoks scale	Leaves	Root	Stem	Spike	Grain
Seedling	First leaf through coleoptile	1	10	x	x
Three leaves	3 leaves unfolded		13		x
Three tillers	Main shoot and 3 tillers		23	x
Spike at 1 cm	Pseudostem erection	5	30			x
Two nodes	2nd detectable node	7	32			x	x
Meiosis	Flag leaf ligule and collar visible	9	39		x		x
Anthesis	1/2 of flowering complete		65			x	x
2 DAAs
(50°C.days)"	Kernel (caryopsis) watery ripe		71	x				x
14 DAAs
(350°C.days)"	Medium Milk		75					x
30 DAAs
(700°C.days)"	Soft dough		85					x

The computation of the expression values for the main Chinese Spring dataset available in POTAGE is described in this paper. The relevant paragraph is included below:
To obtain gene expression information for genes located in the QTL region we used the PopSeq map for bread wheat together with the high confidence (HC) gene predictions described in IWGSC (2014) and the publicly available RNA-Seq data for cv. Chinese Spring. This dataset covers five different organs (root, leaf, stem, spike, grain) at three developmental stages, each in two replicates. We used the HC gene predictions, version 2.1 as reference for mapping the RNA-Seq reads. The reference was prepared by extracting the genomic sequence for each of the predicted genes with up to 2kb upstream and downstream bases whenever available from the corresponding IWGSC-CSS contig. The coordinates in the corresponding GTF/GFF transcript annotations file were adjusted accordingly. RNA-Seq reads were quality-, adapter- and length-trimmed using Trimmomatic (Bolger et al., 2014), version 0.30, with a custom list of adapter sequences and the following settings: ‘ILLUMINACLIP:adapters.fa:1:6:6 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:6 MINLEN:60’. After indexing the reference using Bowtie2 (Langmead and Salzberg, 2012) version 2.2.1, trimmed reads were aligned to the reference using TopHat (Kim et al., 2013) version 2.2.1, not allowing any mismatches or indels. Paired reads were required to map concordantly (--no-discordant setting) to the same (--no-mixed setting) reference sequence. BAM files for the biological replicates were merged, apart from a single tissue/stage sample (spike_Z39) where only one sample was available. Expression was quantified by Cufflinks (Roberts et al., 2011) version 2.1.1 utilizing (through the --GTF option) the adjusted version of the reference transcript annotations provided with the HC gene predictions. The remaining settings were left at their defaults, except for: --max-multiread-fraction 1, --frag-len-std-dev 50 --max-intron-length 5 000. FPKMs (fragments per kilobase of exon per million fragments mapped) per gene (rather than per isoform) were extracted and aggregated in tabular form.

POTAGE was last updated in November 2017

How to cite

If you use POTAGE, please cite the following paper:

https://www.nature.com/articles/s41598-017-14591-7

doi:10.1038/s41598-017-14591-7

Words of caution

The expression values are computed per MIPS gene prediction and to a large extent rely on the correctness of the predictions. Some gene predictions are partial due to the underlying assemblies being highly fragmented.
Occasionally, two separate genes may fall under one Traes ID and in such cases the associated annotation and expression values may be quite misleading.
POPSEQ map does not anchor all the gene-carrying contigs, also note that

Number of contigs/cM interval dependent on recombination frequency
The resolution of a genetic map is dependent on the population size

Helpsheet 1 - Finding genes by gene ID, contig ID

Click on "Search by identifier" icon in the dock-menu at the top of the page
Paste or type in your identifier, POTAGE recognizes two types of identifiers
1. MIPS HCS gene predictions' identifiers, e.g. Traes_2AS_C3EF3A27F
2. IWGSC CSS identifiers, e.g. 5BS_1598873
Click 'Search'

Helpsheet 2 - Finding genes by sequence alignment (BLASTn)

Click on "Search by sequence" icon in the dock-menu at the top of the page
Upload a FASTA file or paste one or more sequences in FASTA format
Once the job has finished you will be redirected to

"Results - per query" tab if you submitted one sequence (skip to 5)
"Results - summary" tab if you submitted more than one sequence

In the former case, selecting one of the sequences takes you to the results for that particular query

In the "Results - per query" tab each row in the table represents a hit

When a row (hit) is selected, POTAGE tries to locate the underlying contig. If found, the contig will be highlighted in one of the two available output tables. Otherwise an appropriate message will be displayed.

If, as in this case, the contig holds more than one gene you can identify the correct gene by comparing the coordinates of your hit in "Results - per query" table with "From" and "To" coordinates of the gene on that contig in the main table.
Please note that if a CSS contig carries more than one HCS gene prediction it will be present in the the main output table multiple times on consecutive positions. Consequently, if the gene/contig is the last one on a page it is best to investigate the next page as it may contain another gene on the same contig (also highlighted).

Helpsheet 3 - Identifying candidate genes within a cM range on a chromosome

Select your chromosome of interest form the dock-menu at the top of the page

This will populate the main table below, listing HCS gene predictions identified on this chromosome.
Next, set appropriate cM range at the top of the main table
Depending on the number of gene predictions present in the selected range, you may be able to browse through their expression profiles by using one of the buttons present for each gene in the "Expression" column of the main table.
- View displays expression chart(s) in a dialog box.
- Adds the expression chart(s) to the "Expression charts display" panel
- Redirects you to view the expression values for the relevant gene on on WheatExp: An Expression Database for Polyploid Wheat
You can also select one or more genes and collectively add those to the "Expression charts display" by clicking in the bottom left corner of the main table

Helpsheet 4 - Rules of thumb for interpreting expression values

Various limitations of the underlying datasets and the methods used mean that the expression values available in POTAGE should be treated with caution. Here are some examples of how different FPKM values could be interpreted.

Low abundant transcript (Myb TF)

Frequently Asked Questions (FAQ)

Why is a gene displayed on chromosome 3A even though the identifier indicates it comes from chromosome 1AL?: Both the chromosome survey sequences (CSS) and POPSEQ can allocate a gene/contig incorrectly, for consistency we rely on POPSEQ's allocation of contigs to chromosomes wherever such information is available, in the remaining cases we fall back to CSS identifiers.
Why is my contig/gene of interest lacking a cM value in the main table?: POPSEQ map does not capture all of the CSS contigs, in fact only about half of contigs for which HC gene predictions are available have been anchored. These genes are appended to the end of the table for their respective chromosome.
How were the expression values generated?: The computation of the expression values for the main Chinese Spring dataset available in POTAGE is described in this paper starting from the second sentence under "Gene identification and RNA-Seq data" subsection.
Can I run POTAGE locally on my own hardware?: Yes, we have developed POTAGE with this in mind. By far the easiest way to do this is to use our Docker image (crobiad/potage). To get started, you will first need to install the "Docker Engine". Once this is installed, simply follow the Quick Start instructions.

	Expression	Gene IDFilter by Gene ID	From	To	+/-	Contig IDFilter by Contig ID	cM	MIPS Annotation			Rice-derived Annotation
	Expression	Gene IDFilter by Gene ID	From	To	+/-	Contig IDFilter by Contig ID	cM	Hit IDFilter by Hit ID	DescriptionFilter by Description	InterproIDFilter by InterproID	Hit IDFilter by Hit ID	DescriptionFilter by Description
No records found.