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10x Genomics
Chromium Single Cell Gene Expression

Release notes for Cell Ranger 5.0.1 (12/16/2020):

Bug Fixes

Release notes for Cell Ranger 5.0.0 (11/19/2020):

Changes that apply to Gene Expression and Feature Barcode analysis

Changes that apply to Gene Expression, Feature Barcode, and V(D)J analysis

Changes that apply to V(D)J analysis

  1. Cell Ranger 5.0 introduces a new clonotype grouping algorithm that computationally approximates groups of cells which are descendants of a single, fully rearranged common ancestor and infers the germline sequence of the V genes from each individual in the dataset. In previous versions (4.0 and earlier), the algorithm grouped cells based only on the set of productive CDR3 nucleotide sequences. As a consequence, whenever a true clonotype had a CDR3 mutation, the true exact subclonotypes were presented by the algorithm as multiple separate clonotypes. The previous approach to clonotyping in Cell Ranger 4.0 and earlier led to inaccuracies in the B cell clonotypes due to the grouping by unique CDR3 sequence. Additionally, single-chain clonotypes were reported as separate clonotypes, which could lead to both over- and under-estimation of the size of a given clonotype. The new clonotyping algorithm is improved in specificity, sensitivity, and overall accuracy because it accounts for mutations found in the V(D)J transcript and in the V(D)J junction. It also merges single chain clonotypes into the correct fully-paired clonotypes for both T cells and B cells. Additional cell filters are also imposed during clonotyping to improve data quality.
  2. Changes to V(D)J outputs:
    • The following output files are removed in 5.0: consensus.fastq and consensus_annotations.json
    • The following output files are added in 5.0:
      • Contig info binary file, which would be used as an input to aggregate V(D)J samples
      • Donor reference fasta
    • Two new columns are added to the clonotypes.csv file that displays the iNKT/MAIT evidence.
    • The files filtered_contig_annotations.csv, filtered_contig.fasta, filtered_contig.fastq now only contain data from the contigs in cell barcodes that are productive.
    • A number of new fields are added to consensus_annotations.csv: v_start, v_end, v_end_ref, j_start, j_start_ref, j_end, cdr3_start, cdr3_end
  3. The recommended V(D)J reference packages for human and mouse have been updated from version 4.0 to 5.0. The changes to the V(D)J reference sequences are listed below:
    • HUMAN:
      • Replace IGKV2D-40, whose leader sequence appears to be truncated.
      • Delete IGKV2-18, which is probably a pseudogene.
      • Delete IGLV5-48, which is truncated on the right.
      • Delete TRBV21-1, which has multiple frameshifts.
      • Add IGHV4-30-4, which was missing.
      • Add IGKV1-NL1, which was missing.
      • Add IGHV4-38-2, which was missing.
    • MOUSE:
      • Delete TRAV23, which is frame-shifted.
      • Delete the first base of the constant region gene IGHG2B.
      • Make a six-base insertion in IGKV12-89, based on empirical data.
      • Correct IGHV8-9, whose amino acid sequence showed the canonical C at the end of FWR3 as S. This is consistent with 10x data.
      • Add an allele of IGKV2-109, which was missing.
      • Add IGKV4-56, which was missing.
      • Add IGHV1-2, which was missing.
  4. cellranger aggr now aggregates V(D)J data, allowing users to recompute V(D)J clonotype groupings across the combined data.
  5. Soft deprecation of --force-cells in cellranger vdj:
    • Since Cell Ranger 3.1, due to filters in the VDJ assembler, --force-cells in VDJ pipelines did not behave as users would expect it to behave. Users can only apply --force-cells to the number of barcodes passing the combined filters in the assembler.
    • This makes it effectively impossible for users to increase the number of recovered cells. Rather, it is only possible to reduce the number of recovered cells using --force-cells in this context, unlike the behavior in the cellranger count pipeline.
    • Because this specific flag is likely to be misunderstood by users, and is also not highly requested, we are starting to deprecate it. In Cell Ranger 5.0, --force-cells will be available only as an undocumented silent option. This will also allow users who are using this routinely in their workflows to anticipate eventual deprecation.