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Can Multiome ATAC and Multiome GEX libraries be sequenced together?

Question: Can Multiome ATAC and Multiome GEX libraries be sequenced together?

Answer: Multiome ATAC and Multiome GEX libraries have very different read length requirements. Please refer to the Single Cell Multiome ATAC + Gene Expression sequencing requirements page for specifications for each library type.

Currently, our official stance is that pooling of these library types has not been tested and is not supported. Pooling of these libraries is at your own risk

 

How to demultiplex:

If you demultiplex a flow cell that contains both Muiltiome ATAC and GEX sequences, cellranger-arc mkfastq could fail if you are using the default setting. The reason is that the mkfastq wrapper will attempt to determine whether the flow cell is either an ATAC or GEX flow cell (but not both) based on the sequencing configuration. Once it makes the determination, say ATAC, then it will fail to demultiplex the GEX sequences even if a custom --use-bases-mask is specified.

To demultiplex such a flow cell, it is necessary to add a few parameters. Below, I will provide an example. Suppose we have a flow cell with the following sequencing configuration, according to its RunInfo.xml:

<Reads>
  <Read NumCycles="45" Number="1" IsIndexedRead="N" />
  <Read NumCycles="10" Number="2" IsIndexedRead="Y" />
  <Read NumCycles="24" Number="3" IsIndexedRead="Y" />
  <Read NumCycles="89" Number="4" IsIndexedRead="N" />
</Reads>

 

ATAC:

For this particular flow cell, it's possible to run cellranger-arc mkfastq with --use-bases-mask=Y45,I8n*,Y24,Y89 and --filter-single-index to demultiplex the ATAC sequences.

  • Read Number 1 is aligned to the genome, so the entire read is kept.
  • Read Number 2 contains the i7 sample index, which is 8 bases long.
  • Read Number 3 contains the 10x barcode. Depending on the sequencer's chemistry (forward vs. reverse complement), the 16-bp barcode could either be at the beginning or the end of the 24 bp reads. It's best to keep all 24 bases and let the pipeline figure out where the barcode is, because the analysis would fail if you trim the wrong 8 bases off.
  • Read Number 4 is aligned to the genome, so the entire read is kept.

 

GEX:

For this particular flow cell, it's possible to run cellranger-arc mkfastq with --use-bases-mask=Y28n*,I10,I10n*,Y89 and --filter-dual-index to demultiplex the GEX sequences.

  • Read Number 1 contains the 16-bp 10x barcode and 12-bp UMI, totalling 28 bases.
  • Read Number 2 contains the i7 dual-index, which is 10 bases long.
  • Read Number 3 contains the i5 dual-index, which is 10 bases long.
  • Read Number 4 is aligned to the transcriptome, so the entire read is kept.

Disclaimer: This article and code-snippet are provided for instructional purposes only. 10x Genomics does not support or guarantee the code.

 

Products: Single Cell Multiome ATAC + GEX