PDX RNA seq counts

Question

[ChumBucket2024]

Hi everyone,

I am trying to generate counts using PDX RNA paired end sequencing data, and I don't know if my counts have been done correctly. Here are the results:

__no_feature    4086152 
__ambiguous     1018383
__too_low_aQual 355475
__not_aligned   482193
__alignment_not_unique  5079336

For creating the reference genome, I merged the human and mouse reference genome together.

Since both human and mouse reference genomes had the "chr #" naming scheme, I renamed all the mouse chromosomes to "m_chr #".

Then, I merged them like so:

cat GCF_000001405.40_GRCh38.p14_genomic.fna renamed_renamed_GCF_000001635.27_GRCm39_genomic.fna > merged_genome.fna

I also merged their corresponding gtf files in the same way.

I used the hisat2 / htseq-count pipeline. My number of not_aligned and other numbers look very concerning. Does anybody have experience with PDX RNA seq data processing that can provide any input?

Any advice would be greatly appreciated. Thank you

Answer 1

[ChumBucket2024]

Here is the line of code used to generate the counts:

htseq-count --format=bam --stranded=no ${aligned}/sorted_21116XR-03-125_S82_L001.bam ${combined_genome}/combined_gtf.gtf > ${counts}/nonstra    nded_21116XR-03-125_S82_L001.count

[ChumBucket2024]

I also used fastp to QC the reads beforehand.