Introduction to designing an experiment and setting up a plate plan in tidyqpcr
Edward Wallace
April 2022
Source:vignettes/platesetup_vignette.Rmd
platesetup_vignette.Rmd
Overview
This vignette introduces how to set up microwell plates for tidyqpcr analysis. Start here if you are new-ish to the tidyverse or want to see explanations about how to design experiments and plate layouts. For worked examples of tidyqpcr analysis on 96-well and 384-well plates, see:
- Delta Cq 96-well plate qPCR analysis vignette,
vignette("deltacq_96well_vignette", package = "tidyqpcr")
- Primers and Probes Calibration vignette,
vignette("calibration_vignette", package = "tidyqpcr")
- Multifactorial experiment vignette,
vignette("multifactor_vignette", package = "tidyqpcr")
Setting up plates is partly a technical question of how to use functions in tidyqpcr and the tidyverse, but more fundamentally a question about how to design your experiment. We recommend the community-led best-practice MIQE guidelines: how many replicates do you need, and what information do you need to provide to accompany your analysis?
We suggest thinking through the whole experiment first, including what you will measure, how many replicates, and what figures you will want to make. If you plan all the analysis before even starting to grow your biological samples and extract RNA/DNA, then it is easier to avoid mistakes. Also, the steps from cell growth, through nucleic acid extraction and measurement, to finished figures, go much quicker.
This vignette builds from a 12-well “practice plate” up to a 96 well plate for a plausible small RT-qPCR experiment. The key idea is to design one replicate of your experiment in a small rectangle on the plate, then make copies of this small rectangle across the plate for more replicates or more complicated designs. The goal is that after working through this vignette, the plate setup in the Multifactorial vignette will be easier to follow.
This vignette focuses on one primer set per well (for SYBR data), and doesn’t discuss more than one probe per well (for TaqMan data). Please create an issue on tidyqpcr github repository if your data needs aren’t covered here, and we will try to respond to it.
Setup knitr options and load packages
This loads the packages necessary for the rest of the code to run.
Essential information: target_id, sample_id, prep_type
Each well of your plate measures one or more sequence targets in one
DNA/RNA sample. Each sample may be duplicated with different types of
preparation (e.g. no reverse transcriptase enzyme controls). The minimal
information you need to describe your plate is to specify the target(s),
sample, and preparation type for every well.
So, tidyqpcr expects that your plate plan has at a minimum three pieces
of information per well: target_id, sample_id, and prep_type.
target_id should uniquely identify a primer set or primer set/probe combination that you detect in a well. If you are detecting multiple regions of the same gene, or trialing multiple probes or primer sets, you have to give them different target_id names. We chose to name this variable “target_id” to make it clear that this could refer to a primer set, or a primer set/ probe combination, detecting a single target sequence. Note again that the current version of tidyqpcr has been tested on SYBR/intercalating dye data with one primer set per well only.
sample_id should uniquely identify a nucleic acid
sample in your experiment. sample_id can either describe all the
relevant information, for example HeatShock_10min_RepA
,
InputControl_WildType_Rep3
; or provide other unique
identifying information, for example S013
. Functions
including display_plate_qpcr
and
calculate_deltacq_bysampleid
assume that there is a column
called sample_id
, and use it to decide which wells get
analysed together. We discuss below how to add other kinds of
information/metadata to help your analysis.
prep_type is used for different types of nucleic acid preps from the same sample. Negative controls are crucial, either no template (NT) controls, or specifically for RNA-measuring RT-qPCR, the no-reverse transcriptase control that detects DNA contamination, as discussed in the MIQE guidelines. So for RT-qPCR experiments we expect to have prep_types +RT and -RT for each sample, and for primer calibration we would always have a no template control.
Technical replicates are also necessary for qPCR experiments to track the variability. This occurs as multiple wells, each of which has the same combination of target_id, sample_id, and prep_type.
Using rows and columns to make life easier
Technically, tidyqpcr can cope with any combination of target and sample in any well. As long as the information is associated clearly, later analysis will work fine. However, good systematic designs that are interpretable both by people (you) and by the computer are less error prone.
One systematic approach is to have each row measure exactly one target and each column one sample. Or vice versa: one row per sample, one column per target. This has the advantage of being straightforward to load with a multichannel pipette.
tidyqpcr is setup to make it easy to specify column contents with a
colkey
, and row contents with a rowkey
, then
to combine these into a plan for an entire plate or for a sub-region of
a plate.
A Minimal 48-well plate plan
Let’s imagine we are performing a RT-qPCR experiment measuring:
- Primer sets against 4 genes: ACT1, BFG2, CDC19, and DED1.
- Three biological replicates: rep1, rep2, rep3
- Three technical replicates of +RT and one of -RT
We need 4 * 3 * 4 = 48 wells for this experiment. Let’s put this information into 48 wells of a 96-well plate.
Practice version, only a single technical replicate.
Here we use the function tibble
to make the rowkey data
tibble, and the function rep
to repeat the target_id
information enough times to fill the plate. These functions are imported
into tidyqpcr; access their help files directly by ?tibble
and ?rep
from your R session.
We use the built-in constant LETTERS
to label the well
row (well_row
) with letters A through D, like they are
labeled on a standard 96-well plate.
target_id_levels <- c("ACT1", "BFG2", "CDC19", "DED1")
rowkey4 <- tibble(
well_row = LETTERS[1:4],
target_id = target_id_levels
)
print(rowkey4)
## # A tibble: 4 × 2
## well_row target_id
## <chr> <chr>
## 1 A ACT1
## 2 B BFG2
## 3 C CDC19
## 4 D DED1
Similarly, we put the sample information in a tibble for the columns,
including well_col
for the column name
sample_id_levels <- c("rep1", "rep2", "rep3")
prep_type_levels <- "+RT"
colkey3 <- tibble(
well_col = 1:3,
sample_id = sample_id_levels,
prep_type = prep_type_levels
)
print(colkey3)
## # A tibble: 3 × 3
## well_col sample_id prep_type
## <int> <chr> <chr>
## 1 1 rep1 +RT
## 2 2 rep2 +RT
## 3 3 rep3 +RT
To hold the information about a blank plate, with information on both
the row and column for each well, tidyqpcr has the function
create_blank_plate
:
create_blank_plate(well_row = LETTERS[1:4], well_col = 1:3)
## # A tibble: 12 × 3
## well well_row well_col
## <chr> <fct> <fct>
## 1 A1 A 1
## 2 A2 A 2
## 3 A3 A 3
## 4 B1 B 1
## 5 B2 B 2
## 6 B3 B 3
## 7 C1 C 1
## 8 C2 C 2
## 9 C3 C 3
## 10 D1 D 1
## 11 D2 D 2
## 12 D3 D 3
Access help for this also at ?create_blank_plate
. Note
that there are default functions to make 96-well, 384-well, and
1536-well blank plates, or as above you can customise it.
Now we create our 12-well mini-plate, using the
label_plate_rowcol
function to combine information from the
blank plate template, the rowkey, and the column key.
plate_plan12 <- label_plate_rowcol(
plate = create_blank_plate(well_row = LETTERS[1:4], well_col = 1:3),
rowkey = rowkey4,
colkey = colkey3
)
print(plate_plan12)
## # A tibble: 12 × 6
## well well_row well_col sample_id prep_type target_id
## <chr> <fct> <fct> <chr> <chr> <chr>
## 1 A1 A 1 rep1 +RT ACT1
## 2 A2 A 2 rep2 +RT ACT1
## 3 A3 A 3 rep3 +RT ACT1
## 4 B1 B 1 rep1 +RT BFG2
## 5 B2 B 2 rep2 +RT BFG2
## 6 B3 B 3 rep3 +RT BFG2
## 7 C1 C 1 rep1 +RT CDC19
## 8 C2 C 2 rep2 +RT CDC19
## 9 C3 C 3 rep3 +RT CDC19
## 10 D1 D 1 rep1 +RT DED1
## 11 D2 D 2 rep2 +RT DED1
## 12 D3 D 3 rep3 +RT DED1
We visualise this plate plan using the
display_plate_qpcr
function:
display_plate_qpcr(plate_plan12)
Expanding this practice plan to incorporate replicates can be done by taking this little rectangle and making copies across a larger plate. This strategy of making copies of a small rectangle makes it easier to use multichannel pipettes to speed up plate loading. It also means that technical replicates of the same sample are not in adjacent wells on the plate, correcting for some location-specific artefacts of amplification in the qPCR machine. However, if there are row- or column-specific artefacts, this approach does not allow you detect them separately.
Replicate columns for the sample_ids and prep_types
Here we are putting three technical replicates of +RT and one of -RT for each sample. This approach is reliable if DNA contamination from -RT samples would show up in multiple sample/target combinations.
We could achieve these replicates in the plate plan by explicitly
writing out every time as in c("+RT", "+RT", "+RT", "-RT")
,
or we can again use the rep
function. Below, we use
rep("+RT", times = 9)
to make 9 repeats, meaning that the 3
technical replicates of each of 3 +RT samples are next to each other. We
use the concatenate function c
, to arrange that next to the
single replicates of the 3 -RT samples.
sample_id_levels <- c("rep1", "rep2", "rep3")
prep_type_values <- c(rep("+RT", times = 9), rep("-RT", times = 3))
print(prep_type_values)
## [1] "+RT" "+RT" "+RT" "+RT" "+RT" "+RT" "+RT" "+RT" "+RT" "-RT" "-RT" "-RT"
colkey12 <- tibble(
well_col = 1:12,
sample_id = rep(sample_id_levels, times = 4),
prep_type = prep_type_values
)
print(colkey12)
## # A tibble: 12 × 3
## well_col sample_id prep_type
## <int> <chr> <chr>
## 1 1 rep1 +RT
## 2 2 rep2 +RT
## 3 3 rep3 +RT
## 4 4 rep1 +RT
## 5 5 rep2 +RT
## 6 6 rep3 +RT
## 7 7 rep1 +RT
## 8 8 rep2 +RT
## 9 9 rep3 +RT
## 10 10 rep1 -RT
## 11 11 rep2 -RT
## 12 12 rep3 -RT
Putting the 48-well sample together
plate_plan48 <- label_plate_rowcol(
plate = create_blank_plate(well_row = LETTERS[1:4], well_col = 1:12),
rowkey = rowkey4,
colkey = colkey12
)
print(plate_plan48)
## # A tibble: 48 × 6
## well well_row well_col sample_id prep_type target_id
## <chr> <fct> <fct> <chr> <chr> <chr>
## 1 A1 A 1 rep1 +RT ACT1
## 2 A2 A 2 rep2 +RT ACT1
## 3 A3 A 3 rep3 +RT ACT1
## 4 A4 A 4 rep1 +RT ACT1
## 5 A5 A 5 rep2 +RT ACT1
## 6 A6 A 6 rep3 +RT ACT1
## 7 A7 A 7 rep1 +RT ACT1
## 8 A8 A 8 rep2 +RT ACT1
## 9 A9 A 9 rep3 +RT ACT1
## 10 A10 A 10 rep1 -RT ACT1
## # ℹ 38 more rows
We again visualise this plate plan using the
display_plate_qpcr
function
display_plate_qpcr(plate_plan48)
Adding more samples and repeating targets
What if we want to measure more than one condition, beyond replicates? For example, a control treatment compared to a drug treatment, or a change in nutrient conditions? We can achieve this again by extending the “copied rectangle” approach to include the second condition.
Adding experimental conditions
In our example, let us do this explicitly. For the rowkey we can use
the rep
function to measure each target in conditions
Ctrl
and Drug
, repeating each 4 times.
condition_levels <- c("Ctrl", "Drug")
condition_values <- rep(condition_levels, each = 4)
print(condition_values)
## [1] "Ctrl" "Ctrl" "Ctrl" "Ctrl" "Drug" "Drug" "Drug" "Drug"
Repeating target names without repeating yourself
We also use the function rep
to repeat the target_id
information 4 times, to fill the plate. Again, ask for help using
?rep
.
target_id_levels <- c("ACT1", "BFG2", "CDC19", "DED1")
target_id_values <- rep(target_id_levels, times = 2)
print(target_id_values)
## [1] "ACT1" "BFG2" "CDC19" "DED1" "ACT1" "BFG2" "CDC19" "DED1"
Now combine this into a rowkey:
rowkey8 <- tibble(
well_row = LETTERS[1:8],
target_id = target_id_values,
condition = condition_values
)
print(rowkey8)
## # A tibble: 8 × 3
## well_row target_id condition
## <chr> <chr> <chr>
## 1 A ACT1 Ctrl
## 2 B BFG2 Ctrl
## 3 C CDC19 Ctrl
## 4 D DED1 Ctrl
## 5 E ACT1 Drug
## 6 F BFG2 Drug
## 7 G CDC19 Drug
## 8 H DED1 Drug
Recreating the column key
To make this into a plate, we also need a column key. What’s changed
is that, each sample needs to refer both to a condition and to a
biological replicate. If we kept colkey12
from above, then
the variable sample_id
would no longer point uniquely to a
single sample.
biol_rep_levels <- c("rep1", "rep2", "rep3")
colkey12_twocondition <- tibble(
well_col = 1:12,
biol_rep = rep(biol_rep_levels, times = 4),
prep_type = prep_type_values
)
print(colkey12_twocondition)
## # A tibble: 12 × 3
## well_col biol_rep prep_type
## <int> <chr> <chr>
## 1 1 rep1 +RT
## 2 2 rep2 +RT
## 3 3 rep3 +RT
## 4 4 rep1 +RT
## 5 5 rep2 +RT
## 6 6 rep3 +RT
## 7 7 rep1 +RT
## 8 8 rep2 +RT
## 9 9 rep3 +RT
## 10 10 rep1 -RT
## 11 11 rep2 -RT
## 12 12 rep3 -RT
Combining information into a larger plate plan
Now we put this together into a plan for the whole 96-well plate:
plate_plan96_take1 <- label_plate_rowcol(
plate = create_blank_plate(well_row = LETTERS[1:8], well_col = 1:12),
rowkey = rowkey8,
colkey = colkey12_twocondition
)
print(plate_plan96_take1)
## # A tibble: 96 × 7
## well well_row well_col biol_rep prep_type target_id condition
## <chr> <fct> <fct> <chr> <chr> <chr> <chr>
## 1 A1 A 1 rep1 +RT ACT1 Ctrl
## 2 A2 A 2 rep2 +RT ACT1 Ctrl
## 3 A3 A 3 rep3 +RT ACT1 Ctrl
## 4 A4 A 4 rep1 +RT ACT1 Ctrl
## 5 A5 A 5 rep2 +RT ACT1 Ctrl
## 6 A6 A 6 rep3 +RT ACT1 Ctrl
## 7 A7 A 7 rep1 +RT ACT1 Ctrl
## 8 A8 A 8 rep2 +RT ACT1 Ctrl
## 9 A9 A 9 rep3 +RT ACT1 Ctrl
## 10 A10 A 10 rep1 -RT ACT1 Ctrl
## # ℹ 86 more rows
Here we had to change the create_blank_plate
call to
include all 8 rows.
Making sure sample names are present and unique
This plate plan lacks a sample_id
column, however. In
fact in this example some of the sample_id information is in the rowkey
(the condition) and some comes from the column key (the biological
replicate). To unite this information, we will conveniently use the
unite
function from the tidyr package:
plate_plan96 <- label_plate_rowcol(
plate = create_blank_plate(well_row = LETTERS[1:8], well_col = 1:12),
rowkey = rowkey8,
colkey = colkey12_twocondition
) %>%
unite(sample_id, condition, biol_rep, remove = FALSE)
print(plate_plan96)
## # A tibble: 96 × 8
## well well_row well_col sample_id biol_rep prep_type target_id condition
## <chr> <fct> <fct> <chr> <chr> <chr> <chr> <chr>
## 1 A1 A 1 Ctrl_rep1 rep1 +RT ACT1 Ctrl
## 2 A2 A 2 Ctrl_rep2 rep2 +RT ACT1 Ctrl
## 3 A3 A 3 Ctrl_rep3 rep3 +RT ACT1 Ctrl
## 4 A4 A 4 Ctrl_rep1 rep1 +RT ACT1 Ctrl
## 5 A5 A 5 Ctrl_rep2 rep2 +RT ACT1 Ctrl
## 6 A6 A 6 Ctrl_rep3 rep3 +RT ACT1 Ctrl
## 7 A7 A 7 Ctrl_rep1 rep1 +RT ACT1 Ctrl
## 8 A8 A 8 Ctrl_rep2 rep2 +RT ACT1 Ctrl
## 9 A9 A 9 Ctrl_rep3 rep3 +RT ACT1 Ctrl
## 10 A10 A 10 Ctrl_rep1 rep1 -RT ACT1 Ctrl
## # ℹ 86 more rows
Again, check the help file with ?unite
. The line
unite(sample_id, condition, biol_rep, remove = FALSE)
means
that we create a new variable sample_id
from existing
variables condition
and biol_rep
, and
remove = FALSE
means that we keep the original variables in
the table as well. The syntax %>%
from the magrittr
package is a way to chain functions together.
Now we display the plate to check that we have everything in place:
display_plate_qpcr(plate_plan96)
We could print this plate map and take it into the lab as a visual aid for plate loading.
Creating plate layouts with standard designs
Some tidyqpcr functions provide shortcuts to create plate layouts with standard designs:
- create_colkey_6_in_24()
- create_colkey_4diln_2ctrl_in_24()
- create_colkey_6diln_2ctrl_in_24()
- create_rowkey_4_in_16()
- create_rowkey_8_in_16_plain()
These focus on setting up column keys and row keys for 384-well
plates where samples are repeated in blocks of 4, 6 or 8. Some are
specialised for primer calibration, including serial dilution and
control samples. These functions can be adapted for your own needs. For
example, the default levels of prep_type
are relevant for
RT-qPCR, and you would want to change those for plain qPCR or ChIP-qPCR.
Please consult the function documentation for details of the parameters
and outputs, e.g. ?create_colkey_6_in_24
.
For examples of standard layouts in use, see the vignettes:
- Primers and Probes Calibration vignette,
vignette("calibration_vignette", package = "tidyqpcr")
- Multifactorial experiment vignette,
vignette("multifactor_vignette", package = "tidyqpcr")
What information goes in the plate plan, revisited?
The plate plan should contain:
- All the information you need to identify the sample and target/probe/primer set uniquely.
- Everything you might want to plot and compare with outputs.
For example, suppose you are testing multiple primer sets against the
same target, your favourite gene YFG1
, and you have primer
sets A, B, and C. Then you might want a variable called
Gene
with value YFG1
for all of these, in
addition to the variable target_id
with levels
YFG1_A
, YFG1_B
, and YFG1_C
.
This package, tidyqpcr, builds on the flexible approaches available
from the tidyverse family of packages. We presented above an example of
specifying individual parts of information about a sample, then uniting
them with the tidyr function unite
. There’s also an inverse
to that, separate
: for example if you have samples from
three strains grown in two temperatures in timepoints in multiple
biological replicates, you might specify sample_id as
WT_25C_10min_rep1
, and then use
separate(col = sample_id, into = c("strain", "temperature", "time_min", "biol_rep"), remove = FALSE)
to create individual columns with copies of that information. The key is
to be consistent and to make the descriptions both human-readable and
also computer-readable: human-readable for your sanity,
computer-readable so that your analysis runs automatically and
correctly.
The functions unite
and separate
have
visual descriptions on the RStudio
data wrangling cheat sheat. Another useful tidyr function is
crossing
, which creates a table with all combinations of
the variables that you supply, say if you want to measure all strains in
all conditions.
Hints and Tips on Plate setup
- In tidyqpcr we chose to give all variables snake_case names, such as
sample_id
,prep_type
, following the tidyverse style guide. - What if you are loading a 384-well plate with a fixed-spacing
multichannel pipette that loads every second row? If you want to set up
the plate plan to load the same sample_id/target_id in two adjacent
rows, using
rep(sample_id, each = 2)
might help. - If your experiment spans multiple plates, it can be helpful to
re-use
colkey
androwkey
in defining similar plate plans. You could re-use the entire plate plan if the plates are exact replicates, as long as you ensure thesample_id
name includes replicate information and so is unique for the analysis. - If you want to avoid certain rows or columns, change the
well_row
orwell_col
arguments in your blank plates, row keys, and column keys. For examplecreate_blank_plate(well_row = LETTERS[2:7], well_col = 2:11)
creates a blank 96-well plate with outside rows and columns empty. - If you want your data to display in a preferred order, make it a
factor. “Factor” is the name in R for data that has a fixed and known
set of possible values. R calls these possible values, “levels”. A list
of pets might have levels: “cat”, “dog”, and “hamster”. Then your pets
might have the values
c("cat", "cat", "cat", "dog")
. The same goes for target genes, sample ids, and growth conditions. In this vignette we have tried to distinguish between “levels” and “values” in our code examples. To learn more about factors, see the chapter on factors in R For Data Science (Wickham & Grolemund)