If it had not already been known [27] that ABI3 is involved in
root development, such an observation of expression in the
root could guide us to look for phenotypes in the roots of
abi3 mutants more closely.
5. The Relative Mode option allows you to view expression of a
given gene in each sample relative to its expression in a control
sample, and to ascertain whether the gene’s expression is above
or below this level. If it is above, a red color is used, and if it is
below, a blue color is used to color the tissue in question. For
the Developmental Map, this level has been computed as the
median level across all of the tissues displayed. The Relative
Mode is more useful in the case of “challenge” experiments,
where a hormone or chemical has been applied as part of the
experimental design. The control sample in this case would be
the mock treated or untreated control.
6. If a given gene does not map to an ATH1 probe set, then try
using the “Klepikova Atlas,” “Shoot Apex,” “Embryo,”
“Silique,” or “Germination” Data Sources, as these were generated using RNA-seq.
3.4.2 eFP-Seq Browser
RNA-seq analysis can be thought of an extension of long-standing
methods such as ESTs, SAGE, and MPSS (expressed sequence tags,
serial analysis of gene expression, and massively parallel signature
sequencing, respectively) for gene expression analysis. The main
difference is that the overall number of “tags” that are generated
for a given transcript population is far higher due to the efficiency of
next-generation sequencing machines at generating sequences
cheaply, thereby increasing accuracy and sensitivity. The eFP-Seq
Browser (see Note 7), included in the Bio-Analytic Resource for
Plant Biology, is used to visualize this type of data. This tool allows
us to search among 113 RNA-seq data sets used by Araport 11 to
reannotate the Arabidopsis genome, and a collection of data sets
from different organs and developmental stages published in
Klepikova et al. [28]. The eFP-Seq Browser retrieves the number
of reads mapped and display these above the desired Araport
11 gene model.
1. Go to http://bar.utoronto.ca and select “eFP-Seq Browser”
from the BAR’s homepage.
2. Enter your gene of interest’s AGI ID. In our case, this is
“At3g24650” for the ABI3 gene. Click on the “Load Data”
button.
3. Figure 6 depicts the default output of this search. The data is
presented in the same pictographic manner as the eFP Browser.
Additionally, the RNA-seq coverage is presented above the
selected gene model variant. How well a given coverage profile
maps to a gene model variant can help with the discovery of
alternative splicing events.
38
G. Alex Mason et al.
root development, such an observation of expression in the
root could guide us to look for phenotypes in the roots of
abi3 mutants more closely.
5. The Relative Mode option allows you to view expression of a
given gene in each sample relative to its expression in a control
sample, and to ascertain whether the gene’s expression is above
or below this level. If it is above, a red color is used, and if it is
below, a blue color is used to color the tissue in question. For
the Developmental Map, this level has been computed as the
median level across all of the tissues displayed. The Relative
Mode is more useful in the case of “challenge” experiments,
where a hormone or chemical has been applied as part of the
experimental design. The control sample in this case would be
the mock treated or untreated control.
6. If a given gene does not map to an ATH1 probe set, then try
using the “Klepikova Atlas,” “Shoot Apex,” “Embryo,”
“Silique,” or “Germination” Data Sources, as these were generated using RNA-seq.
3.4.2 eFP-Seq Browser
RNA-seq analysis can be thought of an extension of long-standing
methods such as ESTs, SAGE, and MPSS (expressed sequence tags,
serial analysis of gene expression, and massively parallel signature
sequencing, respectively) for gene expression analysis. The main
difference is that the overall number of “tags” that are generated
for a given transcript population is far higher due to the efficiency of
next-generation sequencing machines at generating sequences
cheaply, thereby increasing accuracy and sensitivity. The eFP-Seq
Browser (see Note 7), included in the Bio-Analytic Resource for
Plant Biology, is used to visualize this type of data. This tool allows
us to search among 113 RNA-seq data sets used by Araport 11 to
reannotate the Arabidopsis genome, and a collection of data sets
from different organs and developmental stages published in
Klepikova et al. [28]. The eFP-Seq Browser retrieves the number
of reads mapped and display these above the desired Araport
11 gene model.
1. Go to http://bar.utoronto.ca and select “eFP-Seq Browser”
from the BAR’s homepage.
2. Enter your gene of interest’s AGI ID. In our case, this is
“At3g24650” for the ABI3 gene. Click on the “Load Data”
button.
3. Figure 6 depicts the default output of this search. The data is
presented in the same pictographic manner as the eFP Browser.
Additionally, the RNA-seq coverage is presented above the
selected gene model variant. How well a given coverage profile
maps to a gene model variant can help with the discovery of
alternative splicing events.
38
G. Alex Mason et al.