gravatar for el24

2 hours ago by

USA

Hi all, I am new to bioinformatics, so I was wondering if someone can help me with some issues I have with cellranger. I'm trying to run cellranger count on Drosophila melanogaster data, but I need a transcriptome reference to run it. I use this link to create the transcriptome reference file using genome sequence (FASTA) and gene annotations (GTF). Based on that, in Ensembl, the recommended genome file to download is annotated as "primary assembly." In NCBI, it is "no alternative - analysis set." I couldn't find either of the titles on Ensemble or NCBI. I used a couple of different files (GTF and FASTA) on Flybase or NCBI, but I couldn't create a reference transcriptome using them as I got errors. Then, I tried below files, to create the reference:

ftp.ensemblgenomes.org/pub/metazoa/release46/fasta/drosophila_melanogaster/dna/Drosophila_melanogaster.BDGP6.28.dna.toplevel.fa.gz
ftp.ensembl.org/pub/release-77/gtf/drosophila_melanogaster/Drosophila_melanogaster.BDGP5.77.gtf.gz

I managed to create the reference file, but when I run cellranger count using this reference transcriptome, I get an error for different replicates. To be more specific, the error is "Low Fraction Reads Confidently Mapped To Transcriptome" that says I got "19.0%, but Ideal > 30%. This can indicate the use of the wrong reference transcriptome, a reference transcriptome with overlapping genes, poor library quality, poor sequencing quality, or reads shorter than the recommended minimum. Application performance may be affected."

Could you please tell me where I can find a reference transcriptome or where I can find a better GTF and FASTA files to create the reference myself?
I appreciate your response, thanks!



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