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Copy file name to clipboardExpand all lines: docs/tutorial_two_pass_search.md
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## Perform two-pass search to analyze variant peptides
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## Perform two-pass search to analyze novel peptides and PTMs
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##### [FragPipe](https://fragpipe.nesvilab.org) can be downloaded [here](https://github.com/Nesvilab/FragPipe/releases). Follow the instructions on that same Releases page to launch the program. See [here](https://fragpipe.nesvilab.org/docs/tutorial_fragpipe.html#configure-fragpipe) for help configuring FragPipe.
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FragPipe has the ability to perform the "two-pass search" to detect novel/variant peptides semi-automatically. It performs the following three steps.
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1. Perform the first search to identify the canonical peptides
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2. Generate new "sub mzML" files by excluding the scans matching to the canonical peptides
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3. Perform the second search to identify the novel/variant peptides
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FragPipe has the ability to perform the "two-pass search" to detect novel peptides and PTMs semi-automatically. It performs the following three steps.
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1. Perform the first search to identify the canonical peptides and/or common PTMs
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2. Generate new "sub mzML" files by excluding the scans having good matches
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3. Perform the second search to identify the novel peptides and/or PTMs
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#### Common issues of two-pass search approaches due to the limited high-quality scans in the second search
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1. The spectra are not mass calibrated or the mass calibration does not work well
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1. performs mass calibration in the first search, and then generates sub mzML files using the calibrated spectra
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2. writes the Percolator models (`*_percolator.weights` files) to the result directory after the first search. These models will be used in the second search.
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The two-pass search approach supports the `Default`, `LFQ-MBR`, and TMT-related workflows.
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##### _An alternaive approach to analyze novel/variant peptides_
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FragPipe can also perform group FDR estimation for the peptides with different levels of evidences. Please check [Perform group FDR estimation for different identification groups](https://fragpipe.nesvilab.org/docs/tutorial_group_fdr.html) for details.
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### The two-pass search approach supports the `Default`, `LFQ-MBR`, and TMT-related workflows.
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### Perform two-pass search for the label-free quantification data
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1. Load the `LFQ-MBR` workflow in the `Workflow` tab
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