confusing results

[mars188]

Hello,
I ran GuaCAMOLE as below.

guacamole --kraken_report sample1_cust_c0_k2report.txt --kraken_file sample1 \
--kraken_db kraken2/GuaCAMOLE/custom_db --read_files sample1_pure_1.fastq.gz sample1_pure_2.fastq.gz \
--read_len 150 --fragment_len 617 --threshold 500 --length_correction taxonomic --plot plot.png --level G \
--output sample1_guac_results_G.txt 

And the output looks like this

I guess, I should be using "GuaCAMOLE_estimate" column for taxa abundance, however, this column contains all "nan".

What could be the reason and how to solve this? The run seems to finish fine with some warning along the way.

As you see, the data has high GC content.
Note: I prepated kraken2 database with --no-masking option as instructed in the GuaCAMOLE manual.

I would really appreciate the help to resolve this issue.

[Laurenz0908]

Hello,

Thank you for your interest in our tool! It seems like all genera other than Agrobacterium are being identified as false positives and therefore removed (GuaCAMOLE prints a warning when this happens and assigns NaN to all estimates). This can happen when the reference genomes in the database do not match well to the genomes of the taxa in the sample. Looking at your taxa, it seems this is a soil sample? For environmental samples we have unfortunately also seen that the quality of the reference genomes in RefSeq can be an issue. Could you tell me the percentage of classified reads overall by Kraken2?
And what you could also try: leave out the fragment_len parameter. If the fragment length is not so accurate or quite variable in your sample this could add an additional source for problems.