cellranger error fix GTF

Question

[marco.barr]

Hi everyone,

I'm running CellRanger pipeline to analyze my single-cell data. During the index construction step using mkref, I encounter the following error.

mkref has failed: error building reference package  
 Error while parsing GTF file /home/hg38/Homo_sapiens.GRCh38.112.filtered.gtf
 Invalid contig name encountered on GTF line 6: 1. The FASTA file has contigs:
 ['GL000008.2', 'GL000009.2', 'GL000194.1', 'GL000195.1', 'GL000205.2', 'GL000208.1', 'GL000213.1', 'GL000214.1', 'GL000216.2', 'GL000218.1', 
 'GL000219.1', 'GL000220.1', 'GL000221.1', 'GL000224.1', 'GL000225.1', 'GL000226.1', 'KI270302.1', 'KI270303.1', 'KI270304.1', 'KI270305.1', 
'KI270310.1', 'KI270311.1', 'KI270312.1', 'KI270315.1', 'KI270316.1', 'KI270317.1', 'KI270320.1', 'KI270322.1', 'KI270329.1', 'KI270330.1', 'KI270333.1', 
'KI270334.1', 'KI270335.1', 'KI270336.1', 'KI270337.1', 'KI270338.1', 'KI270340.1', 'KI270362.1', 'KI270363.1', 'KI270364.1', 'KI270366.1', 'KI270371.1', 
'KI270372.1', 'KI270373.1', 'KI270374.1', 'KI270375.1', 'KI270376.1', 'KI270378.1', 'KI270379.1', 'KI270381.1', 'KI270382.1', 'KI270383.1', 'KI270384.1', 
'KI270385.1', 'KI270386.1', 'KI270387.1', 'KI270388.1', 'KI270389.1', 'KI270390.1', 'KI270391.1', 'KI270392.1', 'KI270393.1', 'KI270394.1', 'KI270395.1', 
'KI270396.1', 'KI270411.1', 'KI270412.1', 'KI270414.1', 'KI270417.1', 'KI270418.1', 'KI270419.1', 'KI270420.1', 'KI270422.1', 'KI270423.1', 'KI270424.1', 
'KI270425.1', 'KI270429.1', 'KI270435.1', 'KI270438.1', 'KI270442.1', 'KI270448.1', 'KI270465.1', 'KI270466.1', 'KI270467.1', 'KI270468.1', 'KI270507.1', 
'KI270508.1', 'KI270509.1', 'KI270510.1', 'KI270511.1', 'KI270512.1', 'KI270515.1', 'KI270516.1', 'KI270517.1', 'KI270518.1', 'KI270519.1', 'KI270521.1', 
'KI270522.1', 'KI270528.1', 'KI270529.1', 'KI270530.1', 'KI270538.1', 'KI270539.1', 'KI270544.1', 'KI270548.1', 'KI270579.1', 'KI270580.1', 'KI270581.1', 
'KI270582.1', 'KI270583.1', 'KI270584.1', 'KI270587.1', 'KI270588.1', 'KI270589.1', 'KI270590.1', 'KI270591.1', 'KI270593.1', 'KI270706.1', 'KI270707.1', 
'KI270708.1', 'KI270709.1', 'KI270710.1', 'KI270711.1', 'KI270712.1', 'KI270713.1', 'KI270714.1', 'KI270715.1', 'KI270716.1', 'KI270717.1', 'KI270718.1', 
'KI270719.1', 'KI270720.1', 'KI270721.1', 'KI270722.1', 'KI270723.1', 'KI270724.1', 'KI270725.1', 'KI270726.1', 'KI270727.1', 'KI270728.1', 'KI270729.1', 
'KI270730.1', 'KI270731.1', 'KI270732.1', 'KI270733.1', 'KI270734.1', 'KI270735.1', 'KI270736.1', 'KI270737.1', 'KI270738.1', 'KI270739.1', 'KI270740.1', 
'KI270741.1', 'KI270742.1', 'KI270743.1', 'KI270744.1', 'KI270745.1', 'KI270746.1', 'KI270747.1', 'KI270748.1', 'KI270749.1', 'KI270750.1', 'KI270751.1', 
'KI270752.1', 'KI270753.1', 'KI270754.1', 'KI270755.1', 'KI270756.1', 'KI270757.1', 'chr1', 'chr10', 'chr11', 'chr12', 'chr13', 'chr14', 'chr15', 'chr16', 'chr17', 
'chr18', 'chr19', 'chr2', 'chr20', 'chr21', 'chr22', 'chr3', 'chr4', 'chr5', 'chr6', 'chr7', 'chr8', 'chr9', 'chrM', 'chrX', 'chrY']

Please fix your GTF and start again.

These are the commands I used to generate the filtered file.

cellranger mkgtf \
   /home/hg38/Homo_sapiens.GRCh38.112.gtf \
  /home/hg38/Homo_sapiens.GRCh38.112.filtered.gtf \
 --attribute=gene_biotype:protein_coding

What should I do? Thank you very much for your help.

[GenoMax]

During the index construction step using mkref,

Do you have a pressing reason to roll your own when 10x makes GRCh38 pre-made indexes available here: https://www.10xgenomics.com/support/software/cell-ranger/downloads#reference-downloads

[ljhgege]

hello. do you solve the propblem ?

[ljhgege]

i meet the same question when add other gtf to GRCh38 gtf. I want to quantify TE in single cell RNAseq.

[marco.barr]

Hi, I managed to resolve the issue by modifying the assembly version of the FASTA file in the mkref command. To ensure that the genetic annotations in the GTF file correctly correspond to the sequences in the FASTA file, the contig (or chromosome) names must be identical in both files. Use this for mkref: Homo_sapiens.GRCh38.dna_rm.primary_assembly.fa

Alternatively, to avoid any issues, you can download the precompiled version of the reference genome as previously recommended:

wget https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-GRCh38-2020-A.tar.gz

Hope this help you,

Regards

Marco

Answer 1

[swbarnes2]

Did you look at what's on line 6? Maybe show it to the people whom you hope will help you?