Question: I have used antibody-derived tags for both (1) capturing cell surface protein and (2) multiplexing samples (cell hashing). Can I use Cell Ranger to analyze my data?
Answer: Cell Ranger supports the analysis of cell multiplexing with 10x's CellPlex technology. Cell Ranger's tag calling algorithm is validated for CellPlex and may not be optimal for custom tags. Therefore, using custom tags, for example, antibody-based hashtag oligos for cell multiplexing application is officially not supported by Cell Ranger.
However, Cell Ranger allows the analysis of cell multiplexed data using custom tags (such as TotalSeqA/B/C). If you also used antibody-based hashtag oligos (HTOs) for sample multiplexing, you may use the multi pipeline in Cell Ranger 6+ to analyze your data following the instructions below.
1. Prepare your antibody FASTQ files. Cell Ranger expects two non-identical sets of FASTQ files for cell surface protein capture (Antibody Capture) and cell multiplexing (Multiplexing Capture). However, given that you used the same antibody library for dual purposes (cell surface protein capturing + cell hashing), you will need to make a copy of your antibody FASTQ files and also make a tiny edit (for example, delete the first read) in the copy. Please make sure you delete the same read in R1 and R2 reads FASTQs (I1 and I2 as well, if available).
Here is an example command that you can use to delete the first read (first 4 lines) from each of the copied antibody FASTQs (named below as ABC_S3_L001*.fastq.gz to include all R1,R2,I1,I2 files) in a loop:
for f in ABC_S3_L001*.fastq.gz; do
zcat $f | sed '1,4d' | gzip > ${f/%.fastq.gz/_mod.fastq.gz}
doneThis will create a new set of FASTQs with filenames ending in _mod.fastq.gz. After the command finishes running, then you can delete the copied ABC files so they don't take up unnecessary space.
2. Create two sets of reference CSV files.
a. Antibody feature reference CSV file. Please see more detail on this page: feature reference.
b. Custom CMO reference CSV file. If the antibodies for both cell surface protein capture and cell multiplexing are from the same panel, the format of the CMO reference (specifically the pattern field) will be identical to the antibody feature reference. Here is an example, for a CMO reference for TotalSeqB panel:
id,name,read,pattern,sequence,feature_type HTO1,HTO1,R2,5PNNNNNNNNNN(BC),CTCATTGTAACTCCT,Multiplexing Capture HTO2,HTO2,R2,5PNNNNNNNNNN(BC),CTGGGCAATTACTCG,Multiplexing Capture
3. Create a Multi Config CSV file. For example:
[gene-expression] reference,/path/to/transcriptome
cmo-set,/path/to/CMO_reference.csv #created in step2a
[feature]
reference,/path/to/Antibody_reference.csv #created in step2b
[libraries] fastq_id,fastqs,feature_types gex,/path/to/fastqs-gex,Gene Expression ab,/path/to/fastqs-ab,Antibody Capture hto,/path/to/fastqs-hto,Multiplexing Capture #created in step1
[samples] sample_id,cmo_ids sample1,HTO1 sample2,HTO2
Now you are ready to run Cell Ranger with the config.csv above:
cellranger multi --id=sample1 --csv=/path/to/config.csv
Disclaimer: This article is provided for instructional purposes only. 10x Genomics does not support or guarantee this workaround.
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