Answers.Body: <p>Your problem is caused by using the transcriptome fasta file rather than the genome fasta file. You've already given it transcriptome information with <code>genome_genes.filtered.gtf</code>, it needs the genomic sequence to go along with that.</p>
  
  <p>As an aside, your GTF filtering command is unlikely to be useful unless you have a Gencode or Ensembl GTF for human. Have a look at the <code>gene_biotype</code>s in your file and decide for yourself what (if anything) needs to be filtered. For example, I made a cellranger reference last night for dm3 (the old fruit fly genome) with:</p>
  
  <pre><code>cellranger mkgtf annotation.gtf genes.gtf \
      --attribute=gene_biotype:protein_coding \
      --attribute=gene_biotype:ncRNA \
      --attribute=gene_biotype:snRNA \
      --attribute=gene_biotype:snoRNA \
      --attribute=gene_biotype:pre_miRNA
  </code></pre>
  
  <p>That should be customized for relevance. It's quite possible that your GTF doesn't even have <code>gene_biotype</code> attributes, that's specific to Ensembl I think.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-05-17T08:36:24.113Z
Answers.Text: Indeed, I am not working with Humans on this project (I tried to include a "model-organisms" tag to emphasise this). I included this from the `cellranger` documentation while troubleshooting the issue. To clarify, the reference "genome" is a FASTA with chromosomes named? I have a recent annotation (GTF) for _A. thaliana_ but the reference (FASTA) is by transcripts in this database. An older full-genome reference is available but I'd rather use a recently updated on is possible.
Answers.UserDisplayName: Tom Kelly ケリー・トム
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-05-17T09:08:49.223Z
Answers.Text: Yes, the reference genome is the fasta file containing chromosomal entries. You can get appropriate GTF files from [Ensembl](http://plants.ensembl.org/Arabidopsis_thaliana/Info/Index), a transcriptome fasta file is of no use to you.
Answers.UserDisplayName: Devon Ryan
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-05-17T06:52:57.880Z
Answers.LastActivityDate: 2018-05-17T06:52:57.880Z
Answers.UserDisplayName: Devon Ryan
Body: <p>I'm analysing single-cell RNA-Seq data using the 10X Genomics <a href="https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger" rel="nofollow noreferrer"><code>cellranger</code></a> platform. While they provide reference data for Mouse and Humans, other species require a reference "transcriptome" to be generated with the cellranger <a href="https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/references" rel="nofollow noreferrer"><code>mkref</code></a> tool. However, I've found it to be very temperamental giving errors such as:</p>
  
  <pre><code>gffread genome_GFF3_genes.gff -T -o genome_genes.gtf
  
  cellranger mkgtf genome_genes.gtf genome_genes.filtered.gtf \
                     --attribute=gene_biotype:protein_coding \
                     --attribute=gene_biotype:lincRNA \
                     --attribute=gene_biotype:antisense \
                     --attribute=gene_biotype:IG_LV_gene \
                     --attribute=gene_biotype:IG_V_gene \
                     --attribute=gene_biotype:IG_V_pseudogene \
                     --attribute=gene_biotype:IG_D_gene \
                     --attribute=gene_biotype:IG_J_gene \
                     --attribute=gene_biotype:IG_J_pseudogene \
                     --attribute=gene_biotype:IG_C_gene \
                     --attribute=gene_biotype:IG_C_pseudogene \
                     --attribute=gene_biotype:TR_V_gene \
                     --attribute=gene_biotype:TR_V_pseudogene \
                     --attribute=gene_biotype:TR_D_gene \
                     --attribute=gene_biotype:TR_J_gene \
                     --attribute=gene_biotype:TR_J_pseudogene \
                     --attribute=gene_biotype:TR_C_gene
  
  cellranger mkref --genome="reference-name" \
                   --fasta="transcriptome.fasta" \
                   --genes="genome_genes.filtered.gtf"
  </code></pre>
  
  <blockquote>
    <p>Fatal INPUT FILE error, no valid exon lines in the GTF file: reference_name/genes/genes.gtf
    Solution: check the formatting of the GTF file. Most likely cause is the difference in chromosome naming between GTF and FASTA file.</p>
    
    <p>subprocess.CalledProcessError: Command '['STAR', '--runMode', 'genomeGenerate', '--genomeDir', 'reference_name/star', '--runThreadN', '1', '--genomeFastaFiles', 'reference_name/fasta/genome.fa', '--sjdbGTFfile', 'reference_name/genes/genes.gtf', '--limitGenomeGenerateRAM', '17179869184', '--genomeSAsparseD', '1', '--genomeSAindexNbases', '12', '--genomeChrBinNbits', '10']' returned non-zero exit status 104</p>
    
    <p>subprocess.CalledProcessError: Command '['STAR', '--runMode', 'genomeGenerate', '--genomeDir', 'reference_name/star', '--runThreadN', '1', '--genomeFastaFiles', 'reference_name/fasta/genome.fa', '--sjdbGTFfile', 'reference_name/genes/genes.gtf', '--limitGenomeGenerateRAM', '17179869184', '--genomeSAsparseD', '1', '--genomeSAindexNbases', '12', '--genomeChrBinNbits', '10']' returned non-zero exit status -11</p>
  </blockquote>
  
  <p>What input data is required for the <code>cellranger</code> to construct a reference? What format do the fasta and gtf files need be in? Is it possible to use a "transcriptome" rather than a "genome" as a reference here? For example, I have a transcriptome reference with fasta names <code>&gt;name</code> for each transcript rather than each chromosome.</p>
  
Tags: <scrnaseq><reference-genome><transcriptome><star><cellranger>
Title: What process and input data is required for a cellranger reference transcriptome?
ThreadText: # What process and input data is required for a cellranger reference transcriptome?
  
  (Asked by: username_0 on 2018-05-17)
  
  I'm analysing single-cell RNA-Seq data using the 10X Genomics [`cellranger`](https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) platform. While they provide reference data for Mouse and Humans, other species require a reference "transcriptome" to be generated with the cellranger [`mkref`](https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/references) tool. However, I've found it to be very temperamental giving errors such as:
  
  ```
  gffread genome_GFF3_genes.gff -T -o genome_genes.gtf
  
  cellranger mkgtf genome_genes.gtf genome_genes.filtered.gtf \
                     --attribute=gene_biotype:protein_coding \
                     --attribute=gene_biotype:lincRNA \
                     --attribute=gene_biotype:antisense \
                     --attribute=gene_biotype:IG_LV_gene \
                     --attribute=gene_biotype:IG_V_gene \
                     --attribute=gene_biotype:IG_V_pseudogene \
                     --attribute=gene_biotype:IG_D_gene \
                     --attribute=gene_biotype:IG_J_gene \
                     --attribute=gene_biotype:IG_J_pseudogene \
                     --attribute=gene_biotype:IG_C_gene \
                     --attribute=gene_biotype:IG_C_pseudogene \
                     --attribute=gene_biotype:TR_V_gene \
                     --attribute=gene_biotype:TR_V_pseudogene \
                     --attribute=gene_biotype:TR_D_gene \
                     --attribute=gene_biotype:TR_J_gene \
                     --attribute=gene_biotype:TR_J_pseudogene \
                     --attribute=gene_biotype:TR_C_gene
  
  cellranger mkref --genome="reference-name" \
                   --fasta="transcriptome.fasta" \
                   --genes="genome_genes.filtered.gtf"
  
  ```
  
  > Fatal INPUT FILE error, no valid exon lines in the GTF file: reference\_name/genes/genes.gtf
  > Solution: check the formatting of the GTF file. Most likely cause is the difference in chromosome naming between GTF and FASTA file.
  >
  > subprocess.CalledProcessError: Command '['STAR', '--runMode', 'genomeGenerate', '--genomeDir', 'reference\_name/star', '--runThreadN', '1', '--genomeFastaFiles', 'reference\_name/fasta/genome.fa', '--sjdbGTFfile', 'reference\_name/genes/genes.gtf', '--limitGenomeGenerateRAM', '17179869184', '--genomeSAsparseD', '1', '--genomeSAindexNbases', '12', '--genomeChrBinNbits', '10']' returned non-zero exit status 104
  >
  > subprocess.CalledProcessError: Command '['STAR', '--runMode', 'genomeGenerate', '--genomeDir', 'reference\_name/star', '--runThreadN', '1', '--genomeFastaFiles', 'reference\_name/fasta/genome.fa', '--sjdbGTFfile', 'reference\_name/genes/genes.gtf', '--limitGenomeGenerateRAM', '17179869184', '--genomeSAsparseD', '1', '--genomeSAindexNbases', '12', '--genomeChrBinNbits', '10']' returned non-zero exit status -11
  
  What input data is required for the `cellranger` to construct a reference? What format do the fasta and gtf files need be in? Is it possible to use a "transcriptome" rather than a "genome" as a reference here? For example, I have a transcriptome reference with fasta names `>name` for each transcript rather than each chromosome.
  
  **Tags:** scrnaseq, reference-genome, transcriptome, star, cellranger
  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2018-05-17. Score: 4
  
  Your problem is caused by using the transcriptome fasta file rather than the genome fasta file. You've already given it transcriptome information with `genome_genes.filtered.gtf`, it needs the genomic sequence to go along with that.
  
  As an aside, your GTF filtering command is unlikely to be useful unless you have a Gencode or Ensembl GTF for human. Have a look at the `gene_biotype`s in your file and decide for yourself what (if anything) needs to be filtered. For example, I made a cellranger reference last night for dm3 (the old fruit fly genome) with:
  
  ```
  cellranger mkgtf annotation.gtf genes.gtf \
      --attribute=gene_biotype:protein_coding \
      --attribute=gene_biotype:ncRNA \
      --attribute=gene_biotype:snRNA \
      --attribute=gene_biotype:snoRNA \
      --attribute=gene_biotype:pre_miRNA
  
  ```
  
  That should be customized for relevance. It's quite possible that your GTF doesn't even have `gene_biotype` attributes, that's specific to Ensembl I think.
  
  > **Comments:**
  > **username_0**: Indeed, I am not working with Humans on this project (I tried to include a "model-organisms" tag to emphasise this). I included this from the `cellranger` documentation while troubleshooting the issue. To clarify, the reference "genome" is a FASTA with chromosomes named? I have a recent annotation (GTF) for _A. thaliana_ but the reference (FASTA) is by transcripts in this database. An older full-genome reference is available but I'd rather use a recently updated on is possible.  
  > **username_1**: Yes, the reference genome is the fasta file containing chromosomal entries. You can get appropriate GTF files from [Ensembl](http://plants.ensembl.org/Arabidopsis_thaliana/Info/Index), a transcriptome fasta file is of no use to you.  
  
  ---
  
  