mixcr preset for Fluidigm C1 platform

[malonzm1]

Hi,

What preset should be used with Fluidigm C1 platform?

Thanks and good day.

[Alex-Davydov]

Hi, It depends on the type of chemistry do you use in C1 platform. Could you explain in more details?

[malonzm1]

Hi,

Thanks. Refer to page 3 of https://fluidigm.my.salesforce.com/sfc/p/#700000009DAw/a/4u0000019joV/BB2ZsZ6tu6Yv9jzLBHstJMnCfDNWGqPjG5ZiMhI01i0. Does this help?

[mizraelson]

Hi, According to the protocol, there are two CELL barcodes: one in the R1 and another in the I1. I would recommend obtaining non-demultiplexed data (i.e., not split by Nextera indices) and trying the following below. Note that you will also need the index read I1 fastq file.

mixcr analyze generic-single-cell-gex \
--species hsa \
--tag-pattern "^(CELLROW:N{6})\^(R2:*)\^(CELLCOLUMN:N{10})" \
--set-whitelist CELLROW=file:fluidigmC1-CELLROW-whitelist.txt \
input_R1.fastq.gz \
input_R2.fastq.gz \
input_I1.fastq.gz \
output

I have attached the whitelist for the CELLROW barcode list, which I obtained from the manufacturer's demultiplex script. You can also add a whitelist for the CELLCOLUMN barcode using a similar list of Nextera indices used for your samples.

Alternatively, if your data has already been demultiplexed and you cannot obtain the original non-demultiplexed files, you can use the command below:

mixcr analyze generic-single-cell-gex \
--species hsa \
--tag-pattern "^(CELLROW:N{6})\^(R2:*)" \
--set-whitelist CELLROW=file:fluidigmC1-CELLROW-whitelist.txt \
{{CELLCOLUMN:a}}_R1.fastq.gz \
{{CELLCOLUMN:a}}_R2.fastq.gz \
output

{{CELLCOLUMN:a}} should be placed in the filename pattern where it determines the column (Nextera index).

It's worth noting that, since it's a 3' RNA-seq protocol, your reads most likely will not cover the CDR3 region. This protocol wasn't designed for immune repertoire analysis.

fluidigmC1-CELLROW-whitelist.txt

[malonzm1]

Many thanks!

[malonzm1]

Hi,

Sorry if this has an obvious answer. Re

mixcr analyze generic-single-cell-gex \
--species hsa \
--tag-pattern "^(CELLROW:N{6})\^(R2:*)" \
--set-whitelist CELLROW=file:fluidigmC1-CELLROW-whitelist.txt \
{{CELLCOLUMN:a}}_R1.fastq.gz \
{{CELLCOLUMN:a}}_R2.fastq.gz \
output

why is R1.fastq.gz (containing barcode info) still necessary when the cells have already been demultiplexed?

[mizraelson]

There are two cell barcodes. The cells can be demultiplexed using the CELLCOLUMN (Nextera index), but not by the CELLROW, which is located inside R1. As a result, every pair of files still contains multiple cells.