Custom reference genome for hybride mouse

Question

[ddjima2014]

I haven’t worked with hybrid genome before, I am trying to create a custom genome to align RNA-Seq that was generated from hybrid mouse. Would you please guide me how to create a custom genome for two hybrid mouse? The strains are C57BL and 6NJ. I have aligned to the mouse reference genome (C57BL) and the unique aligned reads percentage is around 65%, this is not good from my experience.

[GenoMax]

Would you please guide me how to create a custom genome for two hybrid mouse?

What exactly are you trying to do here? Is this a hybrid of two known parents (C57BL and 6NJ) ?

Have you checked a few reads that do not map to make sure they are still mouse and not some contamination.

[igor]

I have aligned to the mouse reference genome (C57BL) and the unique aligned reads percentage is around 65%

For RNA-seq, 65% aligned reads is actually within the normal range. That fraction can change substantially depending on the sample prep, such as RNA quality and library kit.

Different strains should not be that different.

Answer 1

[Shalu Jhanwar]

You can directly download the FASTA sequences from C57BL_6NJ_v1 assembly for Mus musculus from here under "Chromosome" tab. After downloading, you can concatenate all the FASTA files into one to generate a reference fasta for alignment of RNA-seq reads. You can use below code for creating single reference fasta:

cat fasta_file1 fasta_file2 fasta_filen > reference fasta

[GenoMax]

While this would create a hybrid genome it would be a simple compilation of two haploid genomes that are still separate so there would be a large amount of duplication of sequences, which will cause issues with almost all reads multi-mapping. If @ssjima2014 is looking at a hybrid of those two parental strains then the hybrid genome will be recombinant of the two parents, so alternating parts will be identical to parental genomes.

Note: It is not clear to me if a genome assembly is available for C57BL_6NJ_v1 hybrid directly at EBI.