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Formatting input files
To use FragPipe-Analyst, user needs to prepare two files. One is a quantification table obtained after running FragPipe and the other one is the metadata. Tables below summarize the file combinations you should use with FragPipe-Analyst. In short, you will need an experimental_annotation.tsv file as metadata alongside the quantification table you generated through FragPipe. experiment_annotation.tsv is similar to FragPipe manifest internally used for FragPipe, but note that experiment_annotation.tsv is a file with header. You will see more examples below.
For protein-level analysis:
| LFQ | DIA | TMT | |
|---|---|---|---|
| Quantification Table | combined_proteins.tsv generated by IonQuant | PG matrix generated by DIA-NN | Protein or Gene reports generated by TMT-I |
| Metadata | experiment_annotation.tsv | experiment_annotation.tsv | experiment_annotation.tsv |
For peptide-level analysis:
| LFQ | DIA | TMT | |
|---|---|---|---|
| Quantification Table | combined_peptides.tsv generated by IonQuant | PR matrix generated by DIA-NN | Peptide report generated by TMT-I |
| Metadata | experiment_annotation.tsv | experiment_annotation.tsv | experiment_annotation.tsv |
For site-level analysis (PTM quantification such as phosphorylation, acetylation, ubiquitination):
| DIA | TMT | |
|---|---|---|
| Quantification Table | DIA single-site report *.tsv | Single-site TMT-I report *.tsv |
| Metadata | experiment_annotation.tsv | experiment_annotation.tsv |
LFQ
Here is an LFQ experiment of tumor (T) and normal (N) samples. It has three replicates for each tumor:
In FragPipe:
| file | experiment | replicate |
|---|---|---|
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_16_37N_1_140729074952.mzML | Sample1 | |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_17_37N_2_140729101310.mzML | Sample2 | |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_18_37N_3_140729122909.mzML | Sample3 | |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_21_12T_1_140729160716.mzML | Sample4 | |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_22_12T_2_140729182320.mzML | Sample5 | |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_23_12T_3_140729203924.mzML | Sample6 | |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_25_27N_1_140730001730.mzML | Sample7 | |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_26_27N_2_140730023333.mzML | Sample8 | |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_27_27N_3_140730044935.mzML | Sample9 | |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_39_3T_1.mzML | Sample10 | |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_40_3T_2.mzML | Sample11 | |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_41_3T_3.mzML | Sample12 |
In FragPipe-Analyst, if user didn’t change anything:
| file | sample | sample_name | condition | replicate |
|---|---|---|---|---|
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_16_37N_1_140729074952.mzML | Sample1 | N37_1 | N | 1 |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_17_37N_2_140729101310.mzML | Sample2 | N37_2 | N | 2 |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_18_37N_3_140729122909.mzML | Sample3 | N37_3 | N | 3 |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_21_12T_1_140729160716.mzML | Sample4 | T12_1 | T | 1 |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_22_12T_2_140729182320.mzML | Sample5 | T12_2 | T | 2 |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_23_12T_3_140729203924.mzML | Sample6 | T12_3 | T | 3 |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_25_27N_1_140730001730.mzML | Sample7 | N27_1 | N | 1 |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_26_27N_2_140730023333.mzML | Sample8 | N27_2 | N | 1 |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_27_27N_3_140730044935.mzML | Sample9 | N27_3 | N | 1 |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_39_3T_1.mzML | Sample10 | T3_1 | T | 1 |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_40_3T_2.mzML | Sample11 | T3_2 | T | 2 |
| /storage/yihsiao/data/PXD002612/convert/QEPlus07252014_41_3T_3.mzML | Sample12 | T3_1 | T | 3 |
sampleis from theexperimentin FragPipe and used for matching samples. User should not change it.conditionis initially parsed fromexperimentin FragPipe, but users could change later.sample_nameis the same assample(experiment) when FragPipe first generated it, but user could edit this as a way to update the visualization. Values in thesample_namealso couldn’t be simply digits like123or456or start with digits.replicatewill be automatically propagated based on what users specified in FragPipe.
Note that since FragPipe-Analyst focuses on differential expression analysis, you should have more than one condition available in the condition column like T/N or Tumor/NAT in the examples mentioned above. Each condition should have at least two samples.
TMT
For TMT, experimental_annotation.txt should contain following columns: plex, channel, sample, replicate, condition, sample name. It will be automatically generated by FragPipe released recently, but don’t upload the file blindly generated, you will need to annotate replicate and condition columns. Here is an example with four different experimental conditions and each condition is with three replicates:
Example 1:
| plex | channel | sample | sample_name | condition | replicate |
|---|---|---|---|---|---|
| Yeast_KO11 | 126 | MET6_1 | MET6_1 | MET6 | 1 |
| Yeast_KO11 | 127N | MET6_2 | MET6_2 | MET6 | 2 |
| Yeast_KO11 | 127C | MET6_3 | MET6_3 | MET6 | 3 |
| Yeast_KO11 | 128N | HIS4_1 | HIS4_1 | HIS4 | 1 |
| Yeast_KO11 | 128C | HIS4_2 | HIS4_2 | HIS4 | 2 |
| Yeast_KO11 | 129N | HIS4_3 | HIS4_3 | HIS4 | 3 |
| Yeast_KO11 | 129C | URA2_1 | URA2_1 | URA2 | 1 |
| Yeast_KO11 | 130N | URA2_2 | URA2_2 | URA2 | 2 |
| Yeast_KO11 | 130C | URA2_3 | URA2_3 | URA2 | 3 |
| Yeast_KO11 | 131N | WT_1 | WT_1 | WT | 1 |
| Yeast_KO11 | 131C | WT_2 | WT_2 | WT | 2 |
Another example shown below is a common experimental desgin with tumor and normal tissue adjacent to the tumor (NAT) samples:
Example 2:
| plex | channel | sample | sample_name | condition | replicate |
|---|---|---|---|---|---|
| TMT_01s | 126 | CPT0079430001 | C3L-00011_N | NAT | 1 |
| TMT_01s | 127N | CPT0023360001 | C3O-00217_N | NAT | 1 |
| TMT_01s | 127C | CPT0023350003 | C3L-00217_T | Tumor | 1 |
| TMT_01s | 128N | CPT0079410003 | C3L-00271_T | Tumor | 1 |
| TMT_01s | 128C | CPT0087040003 | C3L-00121_T | Tumor | 1 |
| TMT_01s | 129N | CPT0077310003 | C3L-00211_T | Tumor | 1 |
| TMT_01s | 129C | CPT0077320001 | C3N-00911_N | NAT | 1 |
| TMT_01s | 130N | CPT0087050003 | C3L-00011_N | NAT | 1 |
| TMT_01s | 130C | CPT0023710001 | C3L-00011_T_1 | Tumor | 1 |
| TMT_01s | 131N | pool01 | pool01 | pool | 1 |
| TMT_02s | 126 | NCI7-1 | NCI7_rep1 | QC | 1 |
| TMT_02s | 127N | CPT0078840001 | C3L-01011_N | NAT | 1 |
| TMT_02s | 127C | CPT0075570001 | C3P-05117_T | Tumor | 1 |
| TMT_02s | 128N | CPT0075560003 | C3N-05040_N | NAT | 1 |
| TMT_02s | 128C | CPT0078830003 | C3L-00011_T | Tumor | 1 |
| TMT_02s | 129N | CPT0077490003 | C3N-05168_N | NAT | 1 |
| TMT_02s | 129C | CPT0077500001 | C3N-55688_N | NAT | 1 |
| TMT_02s | 130N | CPT0023690003 | C3P-05111_T | Tumor | 1 |
| TMT_02s | 130C | TPC0023710001 | C3L-00011_T_2 | Tumor | 2 |
| TMT_02s | 131N | pool02 | pool02 | pool | 1 |
See TMT tutorial for more detailed analysis you could achieve through FragPipe-Analyst.
DIA
Here is an example of DIA experiment with 20 samples from CPTAC ccRCC discovery dataset:
In FragPipe, users will annotate files as followed:
| file | experiment | replicate |
|---|---|---|
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00010_T.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00183_NAT.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00103_NAT.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00088_NAT.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00004_T.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00183_T.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00004_NAT.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00096_T.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00097_T.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00026_NAT.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00026_T.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00096_NAT.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00103_T.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00010_NAT.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00079_NAT.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00079_T.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00088_T.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00011_NAT.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00011_T.mzML | ||
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00097_NAT.mzML |
Before using FragPipe-Analyst, users need to annotate condition and replicate columns in experiment_annotation.tsv:
| file | sample | sample_name | condition | replicate |
|---|---|---|---|---|
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00010_T.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00010_T.mzML | C3L-00010_T | Tumor | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00183_NAT.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00183_NAT.mzML | C3L-00183_NAT | NAT | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00103_NAT.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00103_NAT.mzML | C3L-00103_NAT | NAT | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00088_NAT.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00088_NAT.mzML | C3L-00088_NAT | NAT | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00004_T.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00004_T.mzML | C3L-00004_T | Tumor | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00183_T.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00183_T.mzML | C3L-00183_T | Tumor | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00004_NAT.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00004_NAT.mzML | C3L-00004_NAT | NAT | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00096_T.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00096_T.mzML | C3L-00096_T | Tumor | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00097_T.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00097_T.mzML | C3L-00097_T | Tumor | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00026_NAT.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00026_NAT.mzML | C3L-00026_NAT | NAT | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00026_T.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00026_T.mzML | C3L-00026_T | Tumor | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00096_NAT.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00096_NAT.mzML | C3L-00096_NAT | NAT | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00103_T.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00103_T.mzML | C3L-00103_T | Tumor | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00010_NAT.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00010_NAT.mzML | C3L-00010_NAT | NAT | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00079_NAT.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00079_NAT.mzML | C3L-00079_NAT | NAT | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00079_T.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00079_T.mzML | C3L-00079_T | Tumor | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00088_T.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00088_T.mzML | C3L-00088_T | Tumor | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00011_NAT.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00011_NAT.mzML | C3L-00011_NAT | NAT | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00011_T.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00011_T.mzML | C3L-00011_T | Tumor | 1 |
| E:\ccRCC_DIA\DIA_20files_mzML\CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00097_NAT.mzML | CPTAC_CCRCC_W_JHU_20190112_LUMOS_C3L-00097_NAT.mzML | C3L-00097_NAT | NAT | 1 |
Upload your files
Once you have prepared your input files, you can follow instructions listed below to retrieve results:
- Go to FragPipe-Analyst website
- Click the Analysis button on the left
- Choose the Data type based on your experiment:
- LFQ — label-free quantification at protein level (DDA)
- LFQ (peptide) — label-free quantification at peptide level (DDA)
- TMT — TMT isobaric labeling at protein/gene level
- TMT (peptide) — TMT isobaric labeling at peptide level
- TMT (site) — TMT isobaric labeling at PTM site level
- DIA — data-independent acquisition at protein level
- DIA (peptide) — data-independent acquisition at peptide level
- DIA (site) — data-independent acquisition at PTM site level
- Use the file chooser to upload the quantification table and the
experiment_annotation.tsvfile - If you are using LFQ or LFQ (peptide), choose the intensity type: Intensity, MaxLFQ Intensity, or Spectral Count
- Optionally expand Advanced Options to configure normalization, imputation, FDR correction, and DE thresholds
- Click the Run button
Check Interpretation document to understand your results.