How to build genome index file with ERCC (spike-in )

Question

[Mike]

Hi all,

I have GRCh37 fasta sequence & GTF annotation file and ERCC fasta sequence file (but I don’t have ERCC annotation file (GTF)). I want to build genome index file including ERCC sequence using STAR. please help me how to build index file with ERCC?

Thanks

Answer 1

[Devon Ryan]

Cat the fasta files together and then add an entry for each spike-in into the GTF file. Note that you could instead just map against the spike-ins and use samtools idxstats to get the counts for the spike-ins. The multimapping rate within the spike-ins and between the spike-ins and the human genome will be very low, so this will produce accurate enough results.

[Mike]

Thanks Devon, Sorry, how to add entry for each spike-in into the GTF file?

[Devon Ryan]

It's a text file, open it in your editor of choice.

[Mike]

Thanks, Sorry for again, but I don't have annotation information for spike-ins. Can I add only spike-in names ?

[Devon Ryan]

The annotation is the name and entire length, which you can get from the fasta file.