I'd like to extract bacterial sequences from a metagenome where ~99% of contigs are from a host insect. My current protocol minimaps every 16S sequences present in the sample, then blasts all those sequences to create a library of full genomes that I minimap against. I'm using the full metagenome as my reference file. I wasn't sure if that makes a difference, but figured I'd mention it. I ran the following command to retrieve 16S sequences that are present in the sample:
minimap2 All_reads.fastq 16S_reference_library.fasta > 16S_map.paf
Minimap2 retrieves every entry in 16S_reference_library.fasta. It's highly improbable some of those sequences are present, or that 25,000 species are present at all. Are there any parameters I could adjust in Minimap2 that might help with this?
Alternatively, I'm trying to implement Krakenuniq and Centrifuge, but I'm concerned that, because I don't have a reference genome for the host insect, they'll have a very high false positive rate.