BD Rhapsody™ Sequence Analysis Pipeline

Release Notes

v3 BD Rhapsody™ Sequence Analysis Pipeline   |   October 2025

Added

• ATAC:
  • Gene Activity output—new modality in the .Cellismo output file and also a separate MEX output file. Gene activity is a Gene-by-Cell matrix, where counts are number of transposase cut sites in the gene body or 2,000 bases upstream of the gene start position
  • Transcription factor motif output—new modality in the .Cellismo output file and also a separate MEX output file. This is a TFmotif-by-cell matrix, where values are z-scores of the enrichment of each TF motif

• VDJ
  • New assembly algorithm improves speed of this step by up to 23 fold (range 7x-23x), enabling the processing of billions of TCR/BCR reads. Metrics are generally equivalent or slightly better.
  • VDJ only pipeline—able to provide only TCR and/or BCR FASTQs and get a cell call and VDJ results. Sample multiplexing with VDJ only is also supported. VDJ in combination with a mRNA assay is still recommended for better cell calling and identification.

• New pipeline node to downsample data to calculate a sequencing saturation curve and median genes per cell curve, which are output on the pipeline report
• Make Rhapsody Reference tool:
  • Added an optional input for Transcription Factor Motif PFM file
  • Will now filter out readthrough transcripts and genes with only readthrough transcripts. Added optional parameter to turn off this filtering
  • Added optional parameter to filter out Y chromosome Pseudo-Autosomal Regions from Human reference build 38

• Pipeline Report:
  • New Read Flow diagram, showing a sankey diagram of read filtering steps for each library and for each of the RNA and/or ATAC modalities
  • New Sequencing Saturation calculator to enable calculation of required total reads to achieve a target saturation value

Updated

• VDJ
  • _VDJ_perCell.csv file CDR3 columns are updated to use CDR3 junction instead of CDR3 alone, resulting in the inclusion of canonical amino acids
  • _VDJ_perCell.csv file added full length pairing columns
  • New column in AIRR outputs "junction_anchored_aa"—a direct translation of only the CDR3 nucleotide sequence, not influenced by upstream frameshifts
  • Update constant region gene identification to prevent mismatched chain types
  • Removed PyIR wrapper and call IgBlast directly

• Basic putative cell calling algorithm updated to fix several edge cases and get more precise cell calls. Increase in putative cell number of ~1% is typical. Use of the Expected Cell Count parameter is highly encouraged
• Pipeline Report:
  • Various metric alert updates
  • Mean bioproducts per cell added to summary section

• Gene expression _MolsPerCell MEX output now contains Ensembl IDs as well as Gene symbols
• Improved library name determination from FASTQ file names
• More aggressive cleanup of polyA sequence in reads to prevent spurious alignments
• Make Rhapsody Reference tool: Extra Sequence input is now included in the BWA-Mem index
• Seven Bridges CWL: Instance types updated to be more performant, and increase size of instances for ATAC related nodes
• ATAC peak annotation now uses transcript features rather than gene features, which better classifies peaks when a gene has multiple transcription start sites
• .Cellismo output file now contains GTF data for genes
• Dimensionality reduction threshold updates: Below 100,000 cells, both t-SNE and UMAP coordinates are generated. Between 100,000 and 300,000 cells, only UMAP coordinates. Above 300,000 cells, a sub-sample of 300,000 cells will be selected and UMAP coordinates generated.

Fixed

• AlignmentAnalysis node was not getting an early cell count estimate, which could cause downstream node scaling issues
• TCR/BCR node failure when the number of valid TCR or BCR reads exceeded 2,147,483,647 reads
• Pipeline Report error when exact cell count parameter specified
• Pipeline Report error when CITE-seq/AbSeq only datasets are run
• Targeted RNA pipeline did not output a DBEC MEX file
• ATAC pipeline could get stuck in QualCLAlign_ATAC for some reference genomes with large numbers of contigs
• Rare issue where an ATAC peak could exceed the length of the contig on which it resides
• Improved handling chromosome names with unexpected characters
• Failure in GenerateSeurat node when there is only 1 AbSeq input
• Rare failure cause by poor quality read 1 data creating a race condition
• Rare failure in ATAC node caused by incorrect BWA-MEM2 binary selection
• ATAC pipeline failure when more than one ATAC library was present in the pipeline inputs
• ATAC pipeline failure when using sample tags or an "Extra seqs" input
• ATAC pipeline discrepancy in putative cell numbers in different output files.