Using Minimap2 to remove host reads. Unmapped read number is higher than input read number

[s4251484]

Hi there

I am using minimap2 to align the microbiome reads against the host genome, to separate the host reads and to use the unmapped reads for my downstream microbiome analysis.

However my unmapped read number is higher than the input read number.

Below is the commands that I ran:

minimap2 -t 24 -ax map-ont $host $infolder/$filename > $outfolder/$idname.sam

samtools view -@ 24 -f 4 $outfolder/$idname.sam > $outfolder/$idname-nonhost.bam

samtools sort --threads 24 $outfolder/$idname-nonhost.bam > $outfolder/$idname-nonhost-sorted.bam

bamToFastq -i $outfolder/$idname-nonhost-sorted.bam -fq $outfolder/$idname-nonhost.fastq

here is the output determined by Nanoplot

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Sample ID	input # Reads (K)	input Total Bases (Mb)	unmapped # Reads (K)	unmapped Total Bases (Mb)	unmapped  read perct	unmapped  bases perct
3815	1514.504	2114.67753	1836.906	2919.44312	121.29%	138.06%
3816	1815.924	2024.97881	1479.988	2250.97226	81.50%	111.16%

I would appreciate if you could explain why that is so and if there is any way to achieve my plan to separate the host reads while retaining the number of unmapped (potentially microbiome) reads. thank you!