# Get Started

## 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 DIANN | Protein or Gene reports generated by in 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 DIANN | Peptide report generated by in TMT-I |

Metadata | experiment_annotation.tsv | 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 |

`sample`

is from the`experiment`

in FragPipe and used for matching samples. User should not change it.`condition`

is intially parsed from`experiment`

in FragPipe, but users could change later.`sample_name`

is the same as`sample`

(`experiment`

) when FragPipe first generated it, but user could edit this as a way to update the visualization.`replicate`

will be automatically propagated based on what users specified in FragPipe. Note that since FragPipe-Analyst focus 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.

### 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 means data dependent label-free quantification

- Use file chooser to upload files you prepared earlier
- If you are using LFQ, choose the intensity type for the analysis
- Click the Run button

Check Interpretation document to understand your results.