Writing genome metadata JSON file into reference folder. Generating STAR genome index (may take over 8 core hours for a 3Gb genome). Writing genes GTF file into reference folder. Writing genome FASTA file into reference folder. finished successfullyĬreating new reference folder at /Danio.rerio_genome inserting junctions into the genome indices sorting Suffix Array chunks and saving them to disk. Writing new genes GTF file (may take 10 minutes for a 1GB input GTF file). The output looks similar to this: filter GTF with cellranger mkgtf. If you are working on a shared computing environment such as an HPC cluster, submit this as a job to prevent competing with other users for resources. This can take several hours, depending on your system. The following is the command: cellranger mkref \ Now that you have the genome FASTA and filtered GTF files needed, set up the command to run the cellranger mkref pipeline. This will output the file Danio_.gtf, which will be used in the next step. A minimal GTF file only needs to contain exon features for protein coding genes. If you are using a GTF file that does not contain gene_biotype attributes or is missing other entries, don't worry too much there may still be enough information to build a reference. If you are interested in seeing all of the filters used to build references available on our support site, click here. To remove these entries from the GTF, add this filter argument to the mkgtf command: -attribute=gene_biotype:protein_coding (see list of accepted biotypes here). Which reads are considered for UMI counting by Cell Ranger?.See these resources for further information: In the case where reads are flagged as multi-mapped, they are not counted. These entries can cause reads to be flagged as mapped to multiple genes (multi-mapped) because of the overlapping annotations. GTF files can contain entries for non-polyA transcripts that overlap with protein-coding gene models. wget ĭecompress the file with the gunzip command: gunzip Danio_assembly.fa.gz The file is approximately 400 MB and takes several minutes to download, depending on your system.
#How to change cdf files to something readable download#
Paste the URL into the comandline and download it with the wget command: Right-click on the link to copy the address. Download the FASTA file containing all the chromosomes together in the genome, which has primary assembly in the filename. Select the dna/ directory to access the directory with genome files. Next, navigate back to the Ensembl page for Danio rerio and click on Download FASTA to access the FTP site containing several types of FASTA files. wget ĭecompress the file with the gunzip command: gunzip Danio_.gz The file is approximately 20 MB and takes less than a minute to download depending on your system. Right-click the link to copy the address, paste the URL into the command line, and download using the wget command: For more information on the GTF files in Ensembl, read the README file at the FTP site. All species in Ensembl have similar files available to download. This is the GTF annotation file for this species. This takes you to an FTP site with a list of GTF files available. Navigate to the Gene annotation section of the Ensembl website and click on the Download GTF link. The files needed are located on Ensembl (check this page for any reference updates). This tutorial generates a custom reference for the zebrafish, Danio rerio. (See GFF/GTF File Format - Definition and supported options) Note that a GTF file is required, while a GFF file is not supported. If your species of interest is not available from Ensembl, GTF and FASTA files from other sources can also work. The GTF files from Ensembl contain optional tags that make filtering easy. If the species is available from the Ensembl database, we recommend using the files from there. These steps can be found on this page: Build Notes for Reference Packages.įirst, locate the reference genome FASTA and GTF files for your species. This tutorial follows the same steps used to create the 10x Genomics pre-built references for human and mouse. Reference build instructions: Norwegian rat.Reference build instructions: Rhesus macaque.Add exogenous sequences to a custom reference.Add your marker gene to the FASTA and GTF.The following tutorial outlines the steps to build a custom reference using the cellranger mkref pipeline. Researchers can make custom reference genomes for additional species or add custom marker genes of interest to the reference, e.g. Cell Ranger7.0 (latest), printed on Build a Custom Reference (cellranger mkref)ġ0x Genomics provides pre-built references for human and mouse genomes to use with Cell Ranger.