Introduction to proteomics data analysis: robust summarization

This is part of the online course Proteomics Data Analysis 2021 (PDA21)

1 Background

A study on the facultative pathogen Francisella tularensis was conceived by Ramond et al. (2015) [12]. F. tularensis enters the cells of its host by phagocytosis. The authors showed that F. tularensis is arginine deficient and imports arginine from the host cell via an arginine transporter, ArgP, in order to efficiently escape from the phagosome and reach the cytosolic compartment, where it can actively multiply. In their study, they compared the proteome of wild type F. tularensis (WT) to ArgP-gene deleted F. tularensis (knock-out, D8). For this exercise, we use a subset of the F. tularensis dataset where bacterial cultures were grown in biological quadruplicate and each biorep was run in technical triplicate on a nanoRSLC-Q Exactive PLUS instrument. The data were searched with MaxQuant version 1.4.1.2. and are available on the PRIDE repository: PXD001584.

2 Data

library(tidyverse)
library(limma)
library(QFeatures)
library(msqrob2)
library(plotly)

peptidesFile <- "https://raw.githubusercontent.com/statOmics/MSqRobSumPaper/master/Francisella/data/maxquant/peptides.txt"

ecols <- grep(
  "Intensity\\.",
  names(read.delim(peptidesFile))
  )

pe <- readQFeatures(
  table = peptidesFile,
  fnames = 1,
  ecol = ecols,
  name = "peptideRaw", sep="\t")

colnames(pe)

## CharacterList of length 1
## [["peptideRaw"]] Intensity.1WT_20_2h_n3_1 ... Intensity.3D8_20_2h_n5_3

The annotation can be derived from the file name.

colData(pe)$genotype <- substr(colnames(pe[[1]]),12,13) %>%
                          as.factor
colData(pe)$biorep  <- paste(
  substr(colnames(pe[[1]]),12,13),
  substr(colnames(pe[[1]]),21,22),
  sep="_") %>% as.factor

We calculate how many non zero intensities we have per peptide and this is often useful for filtering.

rowData(pe[["peptideRaw"]])$nNonZero <- rowSums(assay(pe[["peptideRaw"]]) > 0)

Because every biorep is assessed in technical triplicate, we will also calculate the number of biorepeats in which each peptide is observed.

rowData(pe[["peptideRaw"]])$nNonZeroBiorep <- apply(
  assay(pe[["peptideRaw"]]),
  1,
  function(intensity)
    colData(pe)$biorep[intensity>0] %>%
    unique %>%
    length)

Peptides with zero intensities are missing peptides and should be represent with a NA value rather than 0.

pe <- zeroIsNA(pe, "peptideRaw") # convert 0 to NA

2.1 Data exploration

49% of all peptide intensities are missing.

3 Preprocessing

This section preforms preprocessing for the peptide data. This include

• log transformation,
• filtering and
• summarisation of the data.

3.1 Log transform the data

pe <- logTransform(pe, base = 2, i = "peptideRaw", name = "peptideLog")

3.2 Filtering

1. Handling overlapping protein groups

In our approach a peptide can map to multiple proteins, as long as there is none of these proteins present in a smaller subgroup.

pe <- filterFeatures(pe,
  ~ Proteins %in% smallestUniqueGroups(rowData(pe[["peptideLog"]])$Proteins)
)

2. Remove reverse sequences (decoys) and contaminants

We now remove the contaminants and peptides that map to decoy sequences.

pe <- filterFeatures(pe, ~ Reverse != "+")
pe <- filterFeatures(pe, ~ Contaminant != "+")

3. Drop peptides that were only identified in a single biorepeat

Note, that in experiments without technical repeats we filter on the number of samples in which a peptide is picked up. Here, we will require that a peptide is picked up in at least two biorepeats.

pe <- filterFeatures(pe, ~ nNonZeroBiorep >= 2)
nrow(pe[["peptideLog"]])

## [1] 7542

We keep 7542 peptides upon filtering.

3.3 Normalize the data using median centering

We normalize the data by substracting the sample median from every intensity for peptide \(p\) in a sample \(i\):

\[y_{ip}^\text{norm} = y_{ip} - \hat\mu_i\]

with \(\hat\mu_i\) the median intensity over all observed peptides in sample \(i\).

pe <- normalize(pe,
  i = "peptideLog",
  name = "peptideNorm",
  method = "center.median"
)

3.4 Explore normalized data

Upon the normalisation the density curves are nicely registered

pe[["peptideNorm"]] %>%
  assay %>%
  as.data.frame() %>%
  gather(sample, intensity) %>%
  mutate(biorep = colData(pe)[sample,"biorep"]) %>%
  ggplot(aes(x = intensity,group = sample,color = biorep)) +
    geom_density()

## Warning: Removed 40413 rows containing non-finite values (stat_density).

We can visualize our data using a Multi Dimensional Scaling plot, eg. as provided by the limma package.

pe[["peptideNorm"]] %>%
  assay %>%
  limma::plotMDS(col = as.numeric(colData(pe)$genotype),label=colData(pe)$biorep)

The first axis in the plot is showing the leading log fold changes (differences on the log scale) between the samples.

3.5 Summarization to protein level

• By default robust summarization is used: fun = MsCoreUtils::robustSummary()

pe <- aggregateFeatures(pe,
  i = "peptideNorm",
  fcol = "Proteins",
  na.rm = TRUE,
  name = "protein")

## Your quantitative and row data contain missing values. Please read the
## relevant section(s) in the aggregateFeatures manual page regarding the
## effects of missing values on data aggregation.

pe[["protein"]]%>%
  assay %>%
  limma::plotMDS(col = as.numeric(colData(pe)$genotype),label=colData(pe)$biorep)

Note that the samples upon robust summarisation show a clear separation according to the genotype in the first dimension of the MDS plot.

4 Data Analysis

4.1 Estimation

We model the protein level expression values using msqrob. By default msqrob2 estimates the model parameters using robust regression.

We will model the data with a different group mean. The group is incoded in the variable genotype of the colData. We will also have to include a random effect for bio-repeat to address the pseudo-replication in the experiment. Indeed, the data from the same bio-repeat will be correlated!

We can specify this model by using a formula with the factor genotype as a fixed effect and as the factor biorep a random effect: formula = ~genotype + (1|biorep).

In the current implementation of msqrob2, you can only work with mixed models if you set the ridge argument ridge=TRUE. The fixed effects are then estimated using ridge regression and random effects can be introduced in the model. Our implementation exploits the link between penalized ridge regression and mixed models. Indeed, by reformulating the fixed effects as random effects ridge regression can be implemented and the ridge penalty is estimated from the data.

Note, that ridge regression only works if two or more slope parameters have to be estimated for the fixed effects. Here, we only have a factor with two levels resulting in an encoding with only one slope parameter so the msqrob function will throw an error.

try(pe <- msqrob(object = pe, i = "protein", formula = ~ genotype + (1|biorep),ridge=TRUE,overwrite=TRUE) )

## Error in msqrobLmer(y = assay(object[[i]]), formula = formula, data = colData(object),  : 
##   The mean model must have more than two parameters for ridge regression.
##               if you really want to adopt ridge regression when your factor has only two levels
##               rerun the function with a formula where you drop the intercept. e.g. ~-1+condition
## 

However, we can also encode ridge regression for the fixed effects directly by specifying the genotype also as a random effect. More information on the link between ridge regression, mixed models and random effect can be found in (Goeminne, Gevaert, and Clement 2016) and (Sticker et al. 2020)

pe <- msqrob(object = pe, i = "protein", formula = ~ (1|genotype) + (1|biorep), ridge=TRUE)

## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular
## boundary (singular) fit: see ?isSingular

4.2 Inference

First, we extract the parameter names of the model by looking at the first model. The models are stored in the row data of the assay under the default name msqrobModels.

getCoef(rowData(pe[["protein"]])$msqrobModels[[1]])

##            (Intercept) (Intercept)biorepD8_n3 (Intercept)biorepD8_n4 
##           3.675785e-02          -2.694535e-01           5.394845e-02 
## (Intercept)biorepD8_n5 (Intercept)biorepWT_n3 (Intercept)biorepWT_n4 
##           8.643411e-02          -1.003137e-01          -3.294403e-03 
## (Intercept)biorepWT_n5  (Intercept)genotypeD8  (Intercept)genotypeWT 
##           2.326791e-01          -8.654787e-15           8.654787e-15

With our encoding we get an estimate for each genotype: (Intercept)genotypeD8 and (Intercept)genotypeWT.

Thus, we assess the contrast ‘(Intercept)genotypeD8 - (Intercept)genotypeWT = 0’ with our statistical test. Note, that specifying this contrast will not work with the default makeContrast function due to the parameter names. So we therefore first

L <- makeContrast("genotypeD8 - genotypeWT=0", parameterNames = c("genotypeWT", "genotypeD8"))

Next, we change the rownames of the matrix L

rownames(L)<-paste0("(Intercept)",rownames(L))
L

##                       genotypeD8 - genotypeWT
## (Intercept)genotypeWT                      -1
## (Intercept)genotypeD8                       1

pe <- hypothesisTest(object = pe, i = "protein", contrast = L)

4.3 Plots

4.3.1 Volcano-plot

volcano <- ggplot(rowData(pe[["protein"]])$`genotypeD8 - genotypeWT`,
                  aes(x = logFC, y = -log10(pval), color = adjPval < 0.05)) +
  geom_point(cex = 2.5) +
  scale_color_manual(values = alpha(c("black", "red"), 0.5)) + theme_minimal()
volcano

Note, that 142 proteins are found to be differentially abundant.

4.3.2 Heatmap

We first select the names of the proteins that were declared signficant.

sigNames <- rowData(pe[["protein"]])$`genotypeD8 - genotypeWT` %>%
  rownames_to_column("protein") %>%
  filter(adjPval<0.05) %>%
  pull(protein)
heatmap(assay(pe[["protein"]])[sigNames, ])

4.3.3 Detail plots

We will make detail plots for the first 10 DE proteins.

maxPlot <- 10
for (protName in sigNames[1:maxPlot])
{
pePlot <- pe[protName, , c("peptideNorm","protein")]
pePlotDf <- data.frame(longFormat(pePlot))
pePlotDf$assay <- factor(pePlotDf$assay,
                        levels = c("peptideNorm", "protein"))
pePlotDf$condition <- as.factor(colData(pePlot)[pePlotDf$colname, "genotype"])

# plotting
p1 <- ggplot(data = pePlotDf,
       aes(x = colname, y = value, group = rowname)) +
    geom_line() +
    geom_point() +
    theme(axis.text.x = element_text(angle = 70, hjust = 1, vjust = 0.5)) +
    facet_grid(~assay) +
    ggtitle(protName)
print(p1)

# plotting 2
p2 <- ggplot(pePlotDf, aes(x = colname, y = value, fill = condition)) +
  geom_boxplot(outlier.shape = NA) +
  geom_point(
    position = position_jitter(width = .1),
    aes(shape = rowname)) +
  scale_shape_manual(values = 1:nrow(pePlotDf)) +
  labs(title = protName, x = "sample", y = "peptide intensity (log2)") +
  theme(axis.text.x = element_text(angle = 70, hjust = 1, vjust = 0.5)) +
  facet_grid(~assay)
print(p2)
}

Note, that the yeast protein is only covered by 3 peptides. Only one peptide is picked up in condition A. This peptide is also only once observed in spike-in condition B. This puts a considerable burden upon the inference and could be avoided by more stringent filtering.

5 Session Info

With respect to reproducibility, it is highly recommended to include a session info in your script so that readers of your output can see your particular setup of R.

sessionInfo()

## R version 4.1.2 (2021-11-01)
## Platform: x86_64-apple-darwin17.0 (64-bit)
## Running under: macOS Big Sur 10.16
## 
## Matrix products: default
## BLAS:   /Library/Frameworks/R.framework/Versions/4.1/Resources/lib/libRblas.0.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/4.1/Resources/lib/libRlapack.dylib
## 
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
## 
## attached base packages:
## [1] stats4    stats     graphics  grDevices utils     datasets  methods  
## [8] base     
## 
## other attached packages:
##  [1] plotly_4.10.0               msqrob2_1.2.0              
##  [3] QFeatures_1.4.0             MultiAssayExperiment_1.20.0
##  [5] SummarizedExperiment_1.24.0 Biobase_2.54.0             
##  [7] GenomicRanges_1.46.1        GenomeInfoDb_1.30.0        
##  [9] IRanges_2.28.0              S4Vectors_0.32.3           
## [11] BiocGenerics_0.40.0         MatrixGenerics_1.6.0       
## [13] matrixStats_0.61.0          limma_3.50.0               
## [15] forcats_0.5.1               stringr_1.4.0              
## [17] dplyr_1.0.7                 purrr_0.3.4                
## [19] readr_2.1.1                 tidyr_1.1.4                
## [21] tibble_3.1.6                ggplot2_3.3.5              
## [23] tidyverse_1.3.1            
## 
## loaded via a namespace (and not attached):
##  [1] nlme_3.1-153            ProtGenerics_1.26.0     bitops_1.0-7           
##  [4] fs_1.5.2                lubridate_1.8.0         httr_1.4.2             
##  [7] tools_4.1.2             backports_1.4.1         utf8_1.2.2             
## [10] R6_2.5.1                DBI_1.1.2               lazyeval_0.2.2         
## [13] colorspace_2.0-2        withr_2.4.3             tidyselect_1.1.1       
## [16] compiler_4.1.2          cli_3.1.0               rvest_1.0.2            
## [19] xml2_1.3.3              DelayedArray_0.20.0     labeling_0.4.2         
## [22] scales_1.1.1            digest_0.6.29           minqa_1.2.4            
## [25] rmarkdown_2.11          XVector_0.34.0          pkgconfig_2.0.3        
## [28] htmltools_0.5.2         lme4_1.1-27.1           highr_0.9              
## [31] dbplyr_2.1.1            fastmap_1.1.0           htmlwidgets_1.5.4      
## [34] rlang_0.4.12            readxl_1.3.1            rstudioapi_0.13        
## [37] farver_2.1.0            jquerylib_0.1.4         generics_0.1.1         
## [40] jsonlite_1.7.3          BiocParallel_1.28.3     RCurl_1.98-1.5         
## [43] magrittr_2.0.1          GenomeInfoDbData_1.2.7  Matrix_1.3-4           
## [46] Rcpp_1.0.8              munsell_0.5.0           fansi_1.0.2            
## [49] MsCoreUtils_1.6.0       lifecycle_1.0.1         stringi_1.7.6          
## [52] yaml_2.2.1              MASS_7.3-54             zlibbioc_1.40.0        
## [55] grid_4.1.2              parallel_4.1.2          crayon_1.4.2           
## [58] lattice_0.20-45         splines_4.1.2           haven_2.4.3            
## [61] hms_1.1.1               knitr_1.37              pillar_1.6.4           
## [64] igraph_1.2.11           boot_1.3-28             codetools_0.2-18       
## [67] reprex_2.0.1            glue_1.6.0              evaluate_0.14          
## [70] data.table_1.14.2       modelr_0.1.8            nloptr_1.2.2.3         
## [73] vctrs_0.3.8             tzdb_0.2.0              cellranger_1.1.0       
## [76] gtable_0.3.0            clue_0.3-60             assertthat_0.2.1       
## [79] xfun_0.29               broom_0.7.11            AnnotationFilter_1.18.0
## [82] viridisLite_0.4.0       cluster_2.1.2           ellipsis_0.3.2

References

Goeminne, L. J., K. Gevaert, and L. Clement. 2016. “Peptide-level Robust Ridge Regression Improves Estimation, Sensitivity, and Specificity in Data-dependent Quantitative Label-free Shotgun Proteomics.” Mol Cell Proteomics 15 (2): 657–68.

Sticker, A., L. Goeminne, L. Martens, and L. Clement. 2020. “Robust Summarization and Inference in Proteome-wide Label-free Quantification.” Mol Cell Proteomics 19 (7): 1209–19.