Quantification using salmon in alignment-based mode after minimap2 run

Question

[Assa Yeroslaviz]

I'm trying to quantify my ONT samples after using minimap2 to align them tho the genome.

The samples were extracted using a direct-RNA protocol and were therefore mapped using the fasta file from the ensembl reporitory, listing the chromosomes, not the transcriptome of the mouse.

REFERENCE='Mmu.GrCm39.fa' # chromosomes 
minimap2 -ax splice -k 14 -uf \
     --secondary=no -G 25000 -t 24 ${REFERENCE} file.fastq > file.sam

Now, the bam file lists the chromosomes in the header.

@HD     VN:1.6  SO:coordinate
@SQ     SN:1    LN:195154279
@SQ     SN:10   LN:130530862
@SQ     SN:11   LN:121973369
@SQ     SN:12   LN:120092757
@SQ     SN:13   LN:120883175
@SQ     SN:14   LN:125139656
@SQ     SN:15   LN:104073951
@SQ     SN:16   LN:98008968
@SQ     SN:17   LN:95294699
...

If I understand it correctly, to run salmon quant I must have a transcriptome as reference.

Does it mean, I have to re-run minimap2 against the mouse transcriptome, Mus_musculus.GRCm39.cdna.all.fa instead?

Are there other quantification workflows without re-runnung the mapping against the transcriptome?

Thanks

Assa

[GenoMax]

In case you were not aware ONT also provides a transcriptome workflow for long reads: https://github.com/epi2me-labs/wf-transcriptomes

Answer 1

[Rob]

As ATpoint suggests, you must align against the transcriptome. Also, the other relevant flags should be set (i.e. --secondary=no will disable reporting secondary alignments, but you want to retain multimapping for the purposes of quantification). Regardless, if you are looking to quantify long read RNA-seq data (ONT, or PacBio reads), then I'd recommend you consider using oarfish (paper here) rather than salmon with the --ont flag. This is because we've designed oarfish from the ground up for long read quantification. Also, oarfish supports directly aligning your raw reads against a target transcriptome (it uses minimap2-rs, a rust wrapper around minimap2 internally, so you don't have to worry about getting all of the flags / settings right yourself).

Answer 2

[ATpoint]

Yes, must strictly be transcriptome. Reference from developer: long read + salmon? (transcript abundance)