tools for 10X scRNA-seq counts

Question

[fuhaolll2]

i want to use 10X scRNA-seq data to analyse alternative last exon ,after mapping i get .bam file ,which software is a good choice to count exon?

Answer 1

[GouthamAtla]

You need to create a custom reference GTF where each of your exon is a gene. So a uniq gene_id and gene_name for each of your exons and then use that GTF in cell-ranger pipeline from the beginning.

In GTF file, the gene_id and gene_name attributes are used to get gene level counts, which basically sums up exon level counts that belong to a same gene. if you want exon level counts, each exon should have its own unique gene_id gene_name attribute.

For example, lets say you have a gene definition ( dummy example):

chr1 dummy  exon          380   401   .   +   0  gene_id "001"; gene_name "aa";
chr1 dummy  exon          501   650   .   +   2  gene_id "001"; gene_name "aa";
chr1 dummy  exon          700   707   .   +   2  gene_id "001"; gene_name "aa";
chr1 dummy  exon  380   382   .   +   0  gene_id "001"; gene_name "aa";
chr1 dummy  exon   708   710   .   +   0  gene_id "001"; gene_name "aa";

That should be changed to:

chr1 dummy  exon          380   401   .   +   0  gene_id "001_ex1"; gene_name "aa_ex1";
chr1 dummy  exon          501   650   .   +   2  gene_id "001_ex2"; gene_name "aa_ex2";
chr1 dummy  exon          700   707   .   +   2  gene_id "001_ex3"; gene_name "aa_ex3";
chr1 dummy  exon  380   382   .   +   0  gene_id "001_ex4"; gene_name "aa_ex4";
chr1 dummy  exon   708   710   .   +   0  gene_id "001_ex5"; gene_name "aa_ex5";

Then instead of getting counts for gene 001 , now you will get counts for each individual exons in your output.

Note: The exons need not to be renamed to 1,2,3,4, etc. They can be given any unique value.

[fuhaolll2]

Thanks , but coding is difficult for me . How could i get the special gtf from normal gtf

[fuhaolll2]

You mean i should change all type "exon“ into “gene" in GTF'3th col? I also cant understand why need a uniq gene_id and gene_name for each of exons. Thanks for you explain

[GouthamAtla]

I updated my answer. Please read about GTF/GFF formats and how gene level counts are obtained by any quantification methods.