Cytof data analysis toolkit with cell population auto-annotation function
ImmCellTyper was developed based on the framework and structure of CyTOF data analysis workflow described by Nowicka et al (CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets), and has a novel in house semi-supervised clustering tool named BinaryClust, which enables automatic classification and annotation of main cell types as well as in-depth interrogation of certain subpopulation of interest. By using k-means, BinaryClust takes advantage of the behaviour of CyTOF markers, which is log normal distributed with zero inflation in most of cases, to separate positive and negative cell populations of each protein marker. Then align the results with user-defined classification matrix, cells can be classified and annotated as different populations.The accuracy and performance of the automatic annotation is comparable to manual gating. Users can further extract a certain population of interest, to perform further clustering and investigation. Moreover, a variety of tools are integrated to support data quality check, batch effect correction, dimension reduction, unsupervised clustering(flowSOM and Rphenograph), and sophisticated statistical testing for multiple group comparison etc.
The package can be installed via running the below commands:
if(!require(devtools, quietly = TRUE))
install.packages("devtools")
devtools::install_github("JingAnyaSun/ImmCellTyper")
ImmCellTyper pipeline requires fcs files(.fcs), sample metadata(.xlsx), panel metadata(.xlsx), and classification matrix(.csv). It is recommended to put all the files needed under the same folder.
- For running the batch evaluation/normalisation session, an example of sample metadata is listed below, which should contain the information of file names('file_name'), sample ids('sample_id'), grouping('condition'), patient ids('patient_id') and batches('batch'). And for the reference/control/anchor sample in each run, it should be labelled as 'Ref' in 'condition' column:
# A tibble: 4 × 5
file_name sample_id condition patient_id batch
<chr> <chr> <chr> <chr> <dbl>
1 A1.fcs HC1_B1 Ref HC1 1
2 A2.fcs HC2_B1 HC HC2 1
3 Run3_A1.fcs HC1_B2 Ref HC1 2
4 Run3_A2.fcs HC2_B2 HC HC2 2
- For running the differential analysis session, an example of sample metadata is as below. Apart from 'file_name', 'sample_id', 'condition', and'patient_id', users can add more grouping information like 'outcome' etc. But remember to specify it correspondingly in 'prepData' function. Batch information is not needed in this metadata file.
# A tibble: 11 × 5
file_name sample_id condition patient_id outcome
<chr> <chr> <chr> <chr> <dbl>
1 AM_Non_JAK2.fcs pt1 Non_JAK2 pt1 1
2 CJ_JAK2.fcs pt2 JAK2 pt2 1
3 FD_Non_JAK2.fcs pt3 Non_JAK2 pt3 2
4 JB_Non_JAK2.fcs pt4 Non_JAK2 pt4 3
5 JY_JAK2.fcs pt5 JAK2 pt5 1
6 KA_JAK2.fcs pt6 JAK2 pt6 2
7 LC_Non_JAK2.fcs pt7 Non_JAK2 pt7 3
8 LDD_Non_JAK2.fcs pt8 Non_JAK2 pt8 1
9 PC_JAK2.fcs pt9 JAK2 pt9 3
10 SBB_JAK2.fcs pt10 JAK2 pt10 3
11 YE_Non_JAK2.fcs pt11 Non_JAK2 pt11 2
The requirement for panel metadata is that it shall comprise the column names of the fcs files('fcs_colname'), protein names('antigen') and the status of markers(marker_class):'type' means phenotyping markers whereas 'state' indicates functional proteins. An example is shown below:
# A tibble: 36 × 3
fcs_colname antigen marker_class
<chr> <chr> <chr>
1 Y89Di CD45 type
2 Pr141Di CD196_CCR6 type
3 Nd142Di CD19 type
4 Nd143Di CD127_IL-7Ra state
5 Nd144Di CD38 type
6 Nd145Di CD33 type
7 Nd146Di IgD type
8 Sm147Di CD11c type
9 Nd148Di CD16 type
10 Sm149Di CD194_CCR4 state
# ℹ 26 more rows
The classification matrix is defined by users to specify the marker expression of each cell type, based on their biological knowledge and gating strategy. This file should be in the format of csv. As exemplified below: + means positively expressed, - means negatively expressed and A means "any". The marker name should be consistent with the panel metadata 'antigen' column.
# A tibble: 7 × 11
`Cell Type` CD14 CD16 CD161 CD19 CD20 CD3 CD4 CD56_NCAM CD8 TCRgd
<chr> <chr> <chr> <chr> <chr> <chr> <chr> <chr> <chr> <chr> <chr>
1 NK Cells - A A - A - A + A A
2 Dendritic Cells - A A - A - A - A A
3 Monocytes + A A - - - A A A A
4 B Cells - - - + A - A A A A
5 T Cells, Gamma Delta - A A - A + A A A +
6 T Cells, CD4 - A A - A + + A - -
7 T Cells, CD8 - A A - A + - A + -
If you have prepared all the required files mentioned above, then you are ready to start the workflow. It is suggested to first do the data quality and batch effects check(please refer to vignettes/Intro_to_batch_exam_corrct), then start the differential analysis(please see vignettes/Intro_to_data_analysis for detailed instructions).
Example dataset used can be downloaded from Zenodo 10.5281/zenodo.7982165.