Hey guys, I have just recently (9 days ago) posted about metagenomic analysis of phages. I hope it is okay, to post again right now, the issue is different this time. I am struggling a bit with deciding which BLAST parameters I should consider and which changes I could make. I did not find much by going through papers.
I have blasted my assembled contigs first with this command: "blastn -db nt_viruses -negative_gilist neg_list.gi -evalue 0.000001 -outfmt "6 qseqid saccver sscinames slen length qcovs qcovhsp pident evalue bitscore" and also without the -negative_gilist parameter.
I have created the .gi list by searching for "environmental" AND "uncultured" in NCBI Nucleotide GenBank and narrowed the results for Viruses and Genomic DNA in order to exclude environmental and uncultured samples since I had a lot of hits like "Caudovirales sp." which basically does not help me much. I figured that this however also excludes some valuable hits that do not appear to be uncultured to me when looking up the Accession numbers. Is this the right way to mimic the option "exclude Uncultured/environmental sample sequences" like you have in online BLAST?
Also I am a little bit baffled about hits that presumably return phages against Escherichia, Klebsiella, Salmonella, Yersinia etc. although I used P. aeruginosa strains as my host in the sample preparation. I also have phages against Stenotrophomonas which I can understand since these are related. But I do not know if I should just remove the hits on other bacterial genera and just keep the Pseudomonas phages or what to do. Normally there should not be a contamination and I also do not have these results in all samples. Some samples are only Pseudomonas phages.