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README.md
analyze_shm.Rmd
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raji.cys
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raji_R12.bin
raji_R12.edge.txt
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raji_R12.shm.txt
raji_R12.txt

README.md

Post-analysis of MIGMAP output

To summarize somatic hypermutations and generate clonotype trees run

java -Xmx8G -jar migmap.jar com.antigenomics.migmap.Analyze -S human -R IGH raji_R12.fastq raji_R12

This folder contains analysis results for IGH repertoire of hypermutating Raji cell line:

  • Reads stored in raji_R12.fastq.gz.. actually, those are not raw reads, but error-corrected assembled consensuses, see MIGEC.
  • Clonotype table and binary output are stored in raji_R12.txt and raji_R12.bin.

Analysis of somatic hypermutation profile

Rmd SHM analysis

Files:

  • raji_R12.shm.txt - mutation table
  • analyze_shm.Rmd - analysis template

The whole analysis template is stored in R markdown format, which means it can be loaded to Rstudio, customized and executed. Detailed explanation of all analysis steps and plots is embedded into the template. An example PDF output is shown in analyze_shm.pdf (click here to view it).

Clonotype tree

Rmd SHM analysis

Files:

  • out.net.txt - network graph
  • out.node.txt - node attributes
  • out.edge.txt - edge attributes

The analysis can be entirely performed in Cytoscape software as follows.

Loading network

Import the network from tab-delimited files generated by MIGMAP

  • Go to File>Import>Network>File.. and import out.net.txt. Specify from and to columns as source and target.

  • Go to File>Import>Table>File.. and import out.node.txt and out.edge.txt. Select Node Table columns and Edge Table columns respectively under Import Data as...

Specify parameter mapping

For nodes:

  • Node size: continuous mapping for freq attribute.
  • Node label: passthrough mapping for cdr3aa attribute.
  • Node label color: discrete mapping for cdr3.code attribute. Then apply (right-click) a Mapping Value Generator. Clonotypes with close cdr3.code values have similar CDR3 sequences.

For edges:

  • Target arrow shape: select an arrow, the graph is directed and built using parsimony principle.
  • Edge color: continuous mapping for replacement.ratio attribute. Note that it is not S:R ratio, but rather number of replacement mutation among all mutations. Only mutations that are present in target node, but not in its parent (i.e. the difference) are counted here.
  • Width: continuous mapping for shm.count.neg attribute. The more hypermutations separate two clonotypes, the smaller is the edge width.

Finalize

Apply an Edge weighted Spring Embedded Layout from the Layout menu, use shm.count.neg as parameter. An example cytoscape file with the network for Raji cell line that can be also used as a template is stored in this folder (raji.cys).

Clonotype network

Under masterment The concept is a network showing each CDR3 with its children (edges are weighted by hypermutation load/S:R ratio) and edges between distinct CDR3 which are likely hypermutations. E.g. a single mutation within "N" region of CDR3 and consequent match of 5 "N" nucleotides between two different CDR3 tells that they are more likely a result of hypermutation than of independent V-D-J recombination.

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