Assemble clones

The assemble command builds a set of clones using alignments obtained with align command in order to extract specific gene regions (e.g. CDR3). The syntax of assemble is the following:

mixcr assemble [options] alignments.vdjca output.clns

The following flowchart shows the pipeline of assemble:

_images/AssembleFlow.svg

This pipeline consists of the following steps:

  1. The assembler sequentially processes records (aligned reads) from input .vdjca file produced by align. On the first step, assembler tries to extract gene feature sequences from aligned reads (called clonal sequence) specified by assemblingFeatures parameter (CDR3 by default); the clonotypes are assembled with respect to clonal sequence. If aligned read does not contain clonal sequence (e.g. CDR3 region), it will be dropped.

  2. If clonal sequence contains at least one nucleotide with low quality (less than badQualityThreshold parameter value), then this record will be deferred for further processing by mapping procedure. If fraction of low quality nucleotides in deferred record is greater than maxBadPointsPercent parameter value, then this record will be finally dropped. Records with clonal sequence containing only good quality nucleotides are used to build core clonotypes by grouping records by equality of clonal sequences (e.g. CDR3). The sequence quality of the resulting core clonotype will be equal to the total of qualities of the assembled reads. Each core clonotype has two main properties: clonal sequence and count — a number of records aggregated by this clonotype.

  3. After the core clonotypes are built, MiXCR runs mapping procedure that processes records deferred on the previous step. Mapping is aimed on rescuing of quantitative information from low quality reads. For this, each deferred record is mapped onto already assembled clonotypes: if there is a fuzzy match, then this record will be aggregated by the corresponding clonotype; in case of several matched clonotypes, a single one will be randomly chosen with weights equal to clonotype counts. If no matches found, the record will be finally dropped.

  4. After clonotypes are assembled by initial assembler and mapper, MiXCR proceeds to clustering. The clustering algorithm tries to find fuzzy matches between clonotypes and organize matched clonotypes in hierarchical tree (cluster), where each child layer is highly similar to its parent but has significantly smaller count. Thus, clonotypes with small counts will be attached to highly similar “parent” clonotypes with significantly greater count. The typical cluster looks as follows:

    _images/Cluster.svg

    After all clusters are built, only their heads are considered as final clones. The maximal depths of cluster, fuzzy matching criteria, relative counts of parent/childs and other parameters can be customized using clusteringStrategy parameters described below.

  5. The final step is to align clonal sequences to reference V,D,J and C genes. Since the assemblingFeatures are different from those used in align, it is necessary to rebuild alignments for clonal sequences. This alignments are built by more accurate aligner (since all hits are known in advance); thus, better alignments will be built for each clonal sequence.

  6. The result is written to the binary output file (.clns) with a comprehensive information about clones.

Command line parameters

The command line options of assemble are the following:

Option Default value Description
-h, --help   Print help message.
-r {file}
--report ...
  Report file name. If this option is not specified, no report file be produced.
-t {numberOfProcessors}
--threads ...
number of available CPU cores Number of processing threads.
-i {indexFile}
--index ...
  Specify file which will store information about particular reads aggreagated by each clone (mapping readId -> cloneId).
-Oparameter=value   Overrides default value of assembler parameter (see next subsection).

All parameters are optional.

Assembler parameters

MiXCR uses a wide range of parameters that controls assembler behaviour. There are some global parameters and parameters organized in groups for each stage of assembling: cloneClusteringParameters and cloneFactoryParameters. Each group of parameters may contain further subgroups of parameters etc. In order to override some parameter value one can use -O followed by fully qualified parameter name and parameter value (e.g. -Ogroup1.group2.parameter=value).

One of the key MiXCR features is ability to assemble clonotypes by sequence of custom gene region (e.g. FR3+CDR3); target clonal sequence can even be disjoint. This region can be specified by assemblingFeatures parameter, as in the following example:

mixcr assemble -OassemblingFeatures="[V5UTR+L1+L2+FR1,FR3+CDR3]" alignments.vdjca output.clns

(note: assemblingFeatures must cover CDR3).

Other global parameters are:

Parameter Default value Description
minimalClonalSequenceLength 12 Minimal length of clonal sequence
badQualityThreshold 20 Minimal value of sequencing quality score: nucleotides with lower quality are considered as “bad”. If sequencing read contains at least one “bad” nucleotide within the target gene region, it will be deferred at initial assembling stage, for further processing by mapper.
maxBadPointsPercent 0.7 Maximal allowed fraction of “bad” points in sequence: if sequence contains more than maxBadPointsPercent “bad” nucleotides, it will be completely dropped and will not be used for further processing by mapper. Sequences with the allowed percent of “bad” points will be mapped to the assembled core clonotypes. Set -OmaxBadPointsPercent=0 in order to completely drop all sequences that contain at least one “bad” nucleotide.
qualityAggregationType Max Algorithm used for aggregation of total clonal sequence quality during assembling of sequencing reads. Possible values: Max (maximal quality across all reads for each position), Min (minimal quality across all reads for each position), Average (average quality across all reads for each position), MiniMax (all letters has the same quality which is the maximum of minimal quality of clonal sequence in each read).
minimalQuality 0 Minimal allowed quality of each nucleotide of assembled clone. If at least one nucleotide in the assembled clone has quality lower than minimalQuality, this clone will be dropped (remember that qualities of reads are aggregated according to selected aggregation strategy during core clonotypes assembly; see qualityAggregationType).
addReadsCountOnClustering false Aggregate cluster counts when assembling final clones: if addReadsCountOnClustering is true, then all children clone counts will be added to the head clone; thus head clone count will be a total of its initial count and counts of all its children. Refers to further clustering strategy (see below). Does not refer to mapping of low quality sequencing reads described above.

One can override these parameters in the following way:

mixcr assemble -ObadQualityThreshold=10 alignments.vdjca output.clns

In order to prevent mapping of low quality reads (filter them off) one can set maxBadPointsPercent to zero:

mixcr assemble -OmaxBadPointsPercent=0 alignments.vdjca output.clns

Separation of clones with same CDR3 (clonal sequence) but different V/J/C genes

Since v1.8 MiXCR can separates clones with equal clonal sequence and different V, J and C (e.g. do distinguish clones with different IG isotype) genes.

To make analysis more robust to sequencing errors there is an additional clustering step to shrink artificial diversity generated by this separation mechanism.

The following criteria are used on this pre-clusterization step: more abondant clone (clone1) absorbs smaller clone (clone2) if clone2.count < clone1.count * maximalPreClusteringRatio (cloneX.count denotes number of reads in corresponding clone)and clone2 contain top V/J/C gene from clone1 in it’s corresponding gene list.

The following parameter control separation behaviour and pre-clusterization:

Parameter Default value Description
maximalPreClusteringRatio 1.0 See conditions for clustering above for more inforamtion.
separateByV false If false clones with equal clonal sequence but different V gene will be merged into single clone.
separateByJ false If false clones with equal clonal sequence but different J gene will be merged into single clone.
separateByC false If false clones with equal clonal sequence but different C gene will be merged into single clone.

Example, in order to separate IG clones by isotypes use the following options:

mixcr assemble -OseparateByC=true alignments.vdjca output.clns

Clustering strategy

Parameters that control clustering procedure are placed in cloneClusteringParameters parameters group which determines the rules for the frequency-based correction of PCR and sequencing errors:

Parameter Default value Description
searchDepth 2 Maximum number of cluster layers (not including head).
allowedMutationsInNRegions 1 Maximum allowed number of mutations in N regions (non-template nucleotides in VD, DJ or VJ junctions): if two fuzzy matched clonal sequences will contain more than allowedMutationsInNRegions mismatches in N-regions, they will not be clustered together (one cannot be a direct child of another).
searchParameters twoMismatchesOrIndels Parameters that control fuzzy match criteria between clones in adjacent layers. Available predefined values: oneMismatch, oneIndel, oneMismatchOrIndel, twoMismatches, twoIndels, twoMismatchesOrIndels, ... , fourMismatchesOrIndels. By default, twoMismatchesOrIndels allows two mismatches or indels (not more than two errors of both types) between two adjacent clones (parent and direct child).
clusteringFilter .specificMutationProbability 1E-3 Probability of a single nucleotide mutation in clonal sequence which has non-hypermutation origin (i.e. PCR or sequencing error). This parameter controls relative counts between two clones in adjacent layers: a smaller clone can be attached to a larger one if its count smaller than count of parent multiplied by (clonalSequenceLength * specificMutationProbability) ^ numberOfMutations.

One can override these parameters in the following way:

mixcr assemble -OcloneClusteringParameters.searchParameters=oneMismatchOrIndel alignments.vdjca output.clns

In order to turn off clustering one should use the following parameters:

mixcr assemble -OcloneClusteringParameters=null alignments.vdjca output.clns