Reference genome fasta file

Question

[synat.keam]

Dear All,

Hope you are well. I am doing alignment using my bulk RNA seq data for differentially expressed genes. Although I am more interested in protein coding genes, I am keen to align all the genes. Therefore, I used Transcript sequence for my fasta file from Genecode. I am wondering whether it is correct one for the purpose of my expriment? Looking forward to hearing from our senior members and expert here.

Kind regards,

Synat

Answer 1

[Dave Carlson]

What tool are you using for your RNA-Seq alignment/mapping? That choice will determine whether you need to use the transcriptome fasta file or the genome fasta file.

• STAR and HISAT2 both require indexed genome fasta files
• Kallisto requires a transcriptome fasta
• Salmon requires a transcriptome fasta but it is recommended that you do a "decoy-aware" mapping that uses both the genome and the transcriptome (see here for more info).

[synat.keam]

Hi Dave,

I am using Kallisto. Therefore, Transcript sequence fasta should be correct? what is your suggestion? Have a good day!

Regards,

Synat,

[Dave Carlson]

Yes, if you're using Kallisto, then you would want to use the transcript fasta file.

[synat.keam]

Hi Dave,

Thank you for your help and guidance!

Have a good day!

Synat,