During the clonotype grouping stage, cell barcodes are placed in groups called clonotypes. Each clonotype consists of all descendants of a single, fully rearranged common ancestor, as approximated computationally. During this process, some cell barcodes are flagged as likely artifacts and filtered out, meaning that they are no longer called as cells.

T cells: The lack of somatic hypermutation (SHM) in T cell receptors (TCRs) yields biological clonotypes that have identical V(D)J transcripts. Technical artifacts (e.g. arising in reverse transcription) can result in the computed clonotypes having isolated differences. These are rare.

B cells: Fully rearranged B cell receptors (BCRs) can undergo SHM, which can increase antigen affinity. Thus for BCRs, V(D)J transcripts in a clonotype can differ at any position, as shown below:

B cell clonotypes can be hard to infer accurately because SHM can introduce numerous mutations. Cell Ranger v5.0 and above accomplishes B cell clonotype grouping by invoking a module for clonal analysis called enclone which simultaneously filters and groups cells into clonotypes.

For clonotype grouping, enclone mainly gets its information from an internally generated Cell Ranger file called all_contig_annotations.json. The relationship between enclone and Cell Ranger is illustrated here:

For each dataset, enclone derives the reference sequence for V genes in the donor's genome (germline sequence) to use as a reference for SHMs.

• For each V segment, one cell from each approximated clonotype is chosen. Approximated clonotypes are not final clonotypes (i.e., those generated as the final step of the clonotype grouping algorithm).
• The distribution of bases in each position on the V segment (excluding the last 15 bases) is determined.
• A V gene position is considered a germline variant if a non reference base is seen in at least 4 approximated clonotypes, comprising at least 25% of the total number of approximated clonotypes.
• This process is repeated for all cells in all the approximated clonotypes.
• The resulting cell-specific "footprint" defines alternative alleles. There is no restriction on the number of possible alternative alleles.

Germline variant assessment for J genes is currently not performed as it does not greatly enhance clonotype specificity.

Cells are placed into groupings called exact subclonotypes if they have identical V(D)J transcripts. Only productive contigs are used. Exact subclonotypes must have the same number of chains. They must also be identical in their V(D)J sequences and constant region gene assignments. Exact subclonotypes are not required to have identical 5' UTRs. Additionally, the algorithm does not test for SHM in the 5' UTR or constant region.

Exact subclonotypes are iteratively merged into clonotypes based on comparing each pair of exact subclonotypes to each other. Two cells with set criteria of shared differences and minimal CDR3 mutations are deemed to be in the same clonotype. Merging criteria are briefly described here. Visit the enclone help page for details.

• Initially, pairs of exact subclonotypes having 2-3 chains are considered for joining together into a clonotype. Later stages of the clonotype grouping algorithm evaluate and merge exact subclonotypes with 1 chain. Exact subclonotypes having 4 chains (putative doublets) are not joined.
• Two exact subclonotypes are merged if a pair of chains has V-J genes and CDR3 segments of identical length.
• Next, shared somatic hypermutations (SHM) in V-J sequence outside the junction regions are identified between different exact subclonotypes. A mutation is shared if the two chains carry the same substitution or indel with respect to the reference sequence (donor reference for V and universal reference for J). Using the donor reference sequences enables the exclusion of shared germline mutations. Visit the enclone help page for details on how the algorithm calculates the probability of these shared mutations occurring by chance.
• Chains that have too many CDR3 mutations are discarded based on a set threshold. CDR3 nucleotide identity of at least 85% is required for exact subclonotype retention.
• Other join criteria may apply and are discussed in the enclone help page under the step "Other join criteria".

During library generation, artifacts can arise by two mechanisms:

(a) Reverse transcription or sequencing can introduce base call errors. These usually occur at bases having low quality scores. Cells with these low quality bases are screened out, typically at a low rate. (b) Gel Beads-in-emulsion (GEMs) may contain material from two or more cells: entire intact cells, cell fragments, or individual mRNA molecules.

Contamination detection is a complex task and is accomplished via multiple heuristic filters. Some barcode filtering happens during the assembly and cell calling stages of Cell Ranger execution. enclone uses Cell Ranger's cell calling information and additionally performs its own barcode and clonotype filtering by employing a series of heuristic tests. Filtering and clonotype grouping happen simultaneously.

Within Cell Ranger, enclone applies these default filters, some of which are recursive:

Visit the enclone website for detailed documentation.

enclone is also available in beta for exploratory evaluation via the use of command line arguments, permitting granular control over clonotyping and filtering heuristics. enclone can also display clonotypes and infer phylogenetic trees.

enclone is open source, unsupported, and separate from Cell Ranger.