Extract Fungal mRNA reda from RNASeq Sample

Question

[SomeOne]

Hello reader,

We have some mRNA seq samples of Macrophage infection analysis with fungal strains. mRNA was extracted from these analysis and sequenced for illumina 150bpX2.

Human macrophages where used for this analysis. and my colleague extracted rna using the whole well of the assay plate. which means that it has both macrophage and fungus mRNA, no idea in what proportions.

QUESTION: i want to extract the reads which are fungal specific. Both Fungal and Human Ref-genomes are available.

In a previous similar query (available-here), i got a suggestion to use bbsplit.sh from BBMap Suit. Which I tried and it worked. Spliting the reads into Human-Ref-Specific-reads.fq.gz and Fungal-Ref-Specific-Reads.fq.gz

ISSUE: bbsplit.sh uses bbmap.sh as a mapping tool at its core which is not a Splice-Aware aligner like STAR2 or HISAT2 which is prefered and mostly used in generat RNASeq analysis for the purpose of alignments.

The idea which i have works like this

1. Map QC-cleaned reads to HUMAN reference genome using Hisat2/STAR
2. Extract all the reads which do not map to human Ref. Using samtools -f 4 flag.

or

1. Map QC-cleaned reads to FUNGAL reference genome using Hisat2/STAR
2. Extract all the reads which do not map to human Ref. Using samtools -f 4 flag.

RESULT: samtools will only extract reads which show no alignment at all to the provided genome.

I need these infection assay sample specific RNA-reads for annotation of genomes.

How should I move forward with this. If you can suggest anyother way which is better that the idea that i have, it will be rally helpful. This data will also be used for RNAseq study but on later stages.

thank you

[GenoMax]

which means that it has both macrophage and fungus mRNA, no idea in what proportions.

I thought we had sorted this issue out in this thread: Extract FUngal mRNA from RNAseq Data

bbmap.sh and a mapping tool at its core which is not a Splice-Aware aligner l

Not correct. As far as I know bbsplit.sh uses bbmap.sh undercover. BTW: bbmap.sh is absolutely a splice aware aligner.

[SomeOne]

Hi. Yes it was solved but i was unsure if bbsplit was splice aware aligner ot not. I tried looking for the documentation but could not find it. Thanks for the clarification.

Sorry there was a typo in line. bbsplit uses bbmap as an aligner.