iCAT: Immune Cells Analysis software Tool
High throughput sequencing of immune cell receptor sequences presents a unique opportunity to inform our understanding of immunological response to infection and how we can detect it. However, the nature of this process requires analysis of massive datasets that requires streamlining. Here we present ICAT, an R-based method for tackling this data and applying statistical analyses to find significant patterns and divergences.
To install devtools, use the command:
Note: iCAT also uses shiny, shinyjs, data.table, ggplot2, DT, hash, and magrittr. However, those packages will be installed if using
install_github from below.
Using an R interface, type:
Graphical Interface Workflow
> library(iCAT) > iCATinteractive()
This will launch a graphical user interface (GUI) for iCAT. The GUI has three tabs, separating major functionalities: training, library, and prediction.
- In the Training tab, enter your negative training samples (naïve, unexposed, uninfected, pre-infection, etc) using the
Individual samples’ sequencing data should be in .tsv format.
Repeat step 1 for positive training samples (exposed, infected, etc.)
Choose if you want to analyze data by:
CDR3 Amino Acid Sequence(TCRs will need the same CDR3 region to be called ‘Identical’)
TCRV-CDR3-TCRJ(TCRs will need the same TCRBV segment, CDR3 region, and TCRJ segment to be called ‘Identical’) Recommended
Nucleic Acid (DNA)(TCRs will need the exact same DNA rearrangements/sequence across TCRBV, CDR3, and TCRJ)
Max p-value, which determines the minimal degree of statistical significance that iCAT will accept as being potentially "associated" with the positive group. Defaults to p < 0.1.
Min Threshold of Public Sequences, which determines the minimum number of training samples a TCR sequence must be observed in to be considered as potentially "associated" with the positive group. Defaults to 1. Recommend setting at 10% of positive training samples. E.g. if there are 30 positive training samples, a recommended minimum threshold is 3.
Once all options are selected click
A progress bar will show on the bottom-right corner to update on the satatus of training. After finishing, the training tab will show some exploratory tables and a figure regarding the training data and the model built, which can all be downloaded to the user's machine. In addition, the library and prediction tab will unlock.
The Library tab displays a table consisting of the "target associated receptor sequences" (TARS), determined to be statistically associated with exposure to the target/agent/pathogen. The table displays each sequence, number of positive and negative training samples the sequence is present in/absent from, and how statistically associated the sequence is to the positive training data (p-value). The table can be downloaded to the user's computer for further analysis using excel, commandline, etc using the download
Table button below the table.
The Prediction tab allows the user to add sequencing data from unknown samples (e.g. not included in the previous training data) for classification as "Positive" or "Negative" and determining the accuracy of the diagnostic assay.
- Use the
Browsebutton to add samples for prediction. Multiple samples may be uploaded simultaneously.
Predict Independent Sample.
A table will appear after analysis is complete. The table displays sample names along with the prediction "Positive" (red)/ "Negative" (blue), and displays the ‘%TARS’: the percent of individual sequences from the sample that are included in the TARS library. The prediction results can be downloaded as a table.
After loading iCAT with
Define the parameters you would like to use for building the model:
> FIELD <- "vGeneName aminoAcid jGeneName" > P_CUTOFF <- 0.1 > MIN_PUBLIC <- 2
Make lists of .tsv Positive and Negative training samples:
> listPos <- tsvDir("path/to/positve/samples/") > listNeg <- tsvDir("path/to/negative/samples/")
optional Collect summary statistics about training samples:
> trnStats(listPos, listNeg, FIELD) #> # Samples # Clonotypes # Unique Sequences #>Negative 9 101942 281336 #>Positive 9 34158 89150
Read in Positive and Negative training samples:
> naive <- readTrn(listNeg, FIELD, "naive") > vaccs <- readTrn(listPos, FIELD, "vacc")
Build a model using the training data:
> mod <- train(naive, vaccs, listNeg, listPos, FIELD, P_CUTOFF, MIN_PUBLIC, NULL)
optional Produce a table estimating the classification accuracy of the model:
optional Produce a figure showing % of TCR associated with positve samples in positive and negative samples:
optional Produce the library of TCR sequences associated with positve samples:
Predict sample(s) exposure based on model:
> pred(mod, vaccs, "path/to/unknown", "unknown-name.tsv", FIELD)