Analysis modules

Table of VDJtools modules

VDJtools software package contains a comprehensive set of immune repertoire post-analysis routines, which are subdivided into several analysis modules. Each module’s section provides command line usage syntax and parameter descriptions for each of the routines, as well as output example and description.

Basic analysis

Summary statistics, spectratyping, etc

  • CalcBasicStats Computes summary statistics for samples: read counts, mean clonotype sizes, number of non-functional clonotypes, etc
  • CalcSegmentUsage Computes Variable (V) and Joining (J) segment usage profiles
  • CalcSpectratype Computes spectratype, the distribution of clonotype abundance by CDR3 sequence length
  • PlotFancySpectratype Plots spectratype explicitly showing top N clonotypes
  • PlotFancyVJUsage Plots the frequency of different V-J pairings
  • PlotSpectratypeV Plots distribution of V segment abundance by resulting CDR3 sequence length

Diversity estimation

Repertoire richness and diversity

Repertoire overlap analysis

Clonotype sharing between samples

Pre-processing

Filtering and resampling

  • Correct Performs a frequency-based erroneous clonotype correction
  • Decontaminate Filters possible cross-sample contaminations in a set of samples
  • DownSample Performs down-sampling, i.e. takes a subset of random reads from sample(s)
  • FilterNonFunctional Filters non-functional clonotypes
  • SelectTop Selects a fixed number of top (most abundant) clonotypes from sample(s)
  • FilterByFrequency Filters clonotypes based on a specified frequency threshold.
  • ApplySampleAsFilter Filters clonotypes that are present in a specified sample from sample(s)
  • FilterBySegment Filters clonotypes according to their V/D/J segment

Operate on clonotype tables

Clonotype table operations

  • PoolSamples Pools clonotypes from several samples together
  • JoinSamples Joins a set of samples and generates clonotype abundance profiles

Annotation

Functional annotation of clonotype tables (antigen specificity, amino acid properties, etc)

Utilities

Some useful utilities

  • FilterMetadata Filters metadata file by values in specified column
  • SplitMetadata Splits metadata file by specified columns
  • Convert Converts from one software format to another
  • RInstall Installs necessary R dependencies

Output

Each routine generates a comprehensive tabular output and some produce optional graphical output. In case of graphical output, the corresponding R script with specified arguments (at the beginning of the script, commented) will be stored to the analysis folder. Thus, user can uncomment the script arguments, modify the script and re-run it. This behavior be disabled by running VDJtools with discard_scripts argument prior to routine name.

By default, all graphical output is generated in PDF format, to generate PNG images use ``--plot-type png option.

When running routines that output clonotype tables consider the following:

  • Joint and pooled samples are stored in VDJtools fomat
  • Samples produced using ScanDatabase (Available only up to v1.0.5, use VDJdb) or Annotation routine are in VDJtools format and include additional annotation columns. Annotation columns are retained when running most of VDJtools routines
  • When loading a joint/pooled sample into VDJtools, clonotype abundance vectors, incidence counts, etc will be treated as clonotype level annotations
  • Annotation columns will not be preserved when joining/pooling annotated samples, a workaround

here will be to use ApplySampleAsFilter routine

Attention

When exporting a table generated by one of VDJtools routines into R use the following command to parse the input correctly:

read.table("some_table.txt", header=T, quote="", sep = "\t")

Common parameters

There are several parameters that are commonly used among analysis routines:

Shorthand Long name Argument Description
-h --help   Brings up the help message for selected routine
-m --metadata path Path to metadata file. Should point to a tab-delimited file with the first two columns containing sample path and sample id respectively, and the remaining columns containing user-specified data. See Metadata section
-u --unweighted   If present as an option and not set, all statistics will be weighted by clonotype frequency
-i --intersect-type string Overlap type, that specifies which clonotype features (CDR3 sequence, V/J segments, hypermutations) will be compared when checking if two clonotypes match. Allowed values: strict,nt,ntV,ntVJ,aa,aaV,aaVJ and aa!nt.
-p --plot   [plotting] Enable plotting for routines that supports it.
  --plot-type <pdf|png> [plotting] Specifies whether to generate a PDF or PNG file. While latter could be easily embedded, PDF plots have superior quality.
-f --factor string [plotting] Name of the sample metadata column that should be treated as factor. If the name contains spaces, the argument should be surrounded with double quotes, e.g. -f "Treatment type"
-n --factor-numeric   [plotting] Treat the factor as numeric?
-l --label string [plotting] Name of the sample metadata column that should be treated as label. If the name contains spaces, the argument should be surrounded with double quotes, e.g. -l "Patient id"
-c --compress path Compress resulting clonotype tables using GZIP.

Overlap type

Some of VDJtools routines require to define clonotype matching strategy when computing clonotype sharing between samples. This parameter is also used when collapsing clonotype tables, e.g. a common situation is when one is interested in estimating the extent of convergent recombination, which is the number of distinct nucleotide CDR3 sequences per one CDR3 amino acid sequence. This requires to collapse clonotype table by identical CDR3aa field.

The list of strategies is defined below.

Shorthand Rule Note
strict CDR3nt (AND) V (AND) J (AND) SHMs Require full match for receptor nucleotide sequence
nt CDR3nt  
ntV CDR3nt (AND) V  
ntVJ CDR3nt (AND) V (AND) J  
aa CDR3aa  
aaV CDR3aa (AND) V  
aaVJ CDR3aa (AND) V (AND) J  
aa!nt CDR3aa (AND)((NOT) CDR3nt ) Removes nearly all contamination bias from overlap results. Should not be used for samples from the same donor/tracking experiments

As somatic hypermutations (SHMs) are currently not supported by VDJtools, strict and ntVJ options are identical. See VDJtools Clonotype specification for details.