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Creating a custom reference with cellranger mkref for EBV B95-8: duplicate gene id

I am trying to create a custom reference for NC_007605.1 following the instructions here.

To do this, I am using the GTF and FASTA files available on NCBI here. Specifically, I am using GCF_002402265.1_ASM240226v1_genomic.gtf and GCF_002402265.1_ASM240226v1_genomic.fna.

However, the command:

cellranger mkref --genome=EBV-N95 --fasta=GCF_002402265.1_ASM240226v1_genomic.fna --genes=GCF_002402265.1_ASM240226v1_genomic.gtf

Fails with error:

Error detected in GTF file: Duplicate Gene ID found in GTF: HHV4_LMP-2A

I guess the error is related to the circular EBV genome. Any clue how to solve this? Or does anyone know an existing reference of the same genome? I am using cellranger 9.0.1.

Thanks!

gtf cellranger mkref
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updated by marco.barr • marco.barr • written by malcapi • malcapi

Hi malcapi,

your intuition might be correct. EBV has a circular genome, but NC_007605.1 is deposited as a linear 171,823 bp sequence. Probably NCBI's annotation represents this as two separate, non-contiguous gene blocks in the GTF that share the same gene_id. It seems to be a known issue if you look here cellranger.

I think, you could modify the GTF to make the contiguous gene_ids unique for instance, by adding a suffix or something like that. Keep in mind that you will then count the two blocks as distinct features in the count matrix. If you need a single count for LMP-2A, you will have to sum them up during post-processing

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written by marco.barr • marco.barr

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