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  • How to parse blastn output

    I have aligned my shotgun metagenomics reads to NCBI eukaryotic reference database using Blastn to evaluate the dietary assessment from fecal samples of black bears. I've got the blastn output as a tabular format (outfmt 6). I am currently trying to see if a PCR bias/PCR duplicates is influencing our results. I want to see if the ratio of unique subjects to unique queries differs depending on enriched/non-enriched samples (this might indicate that it is something about the enrichment process rather than the PCR that changes the results). So, I extracted the information regarding unique queries and unique subject sequences using the following commands:

    for i in $(ls blastn_out_nt/); do cut -f 1 blastn_out_nt/$i | sort | uniq | wc -l >> query; done

    for i in $(ls blastn_out_nt/); do sort -k2,2 blastn_out_nt/$i | cut -f 2,9,10 | uniq | wc -l >> unique_subjects; done

    Need to mention here that the first column in the blastn output​ is query id, the second column is subject id, 9th and 10th columns are the start and end of alignments in the subject. I wanted to verify that the work is error-free and also have an idea about what explains the pattern.

    Here's what I have got:

    Click image for larger version

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    Does a smaller ratio of unique queries and unique subjects potentially indicate that the input fasta sequences were redundant (pcr duplicates) because they hit the same database entry? Also, how I should explain these figures?

    Click image for larger version

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