No classified, neither unclassified sequences in paired fastq sequences

[MGorostidi]

Hey you all!

I'm trying to characterized some sequences of a RNAseq pool with the aim of seeing if there is any bacteria, fungi or plant in them. I'm running the following command and, even if I have tried different parameters for Kraken2, I'm constantly obtaining the result of 0 classified and 0 unclassified sequences. Could it be an error I have done? Or should I assume that there is no any of these species in my sample?

*They are supposed to be non-human sequences, since the 2 fastq files are the unmapped result from STAR algorithm and have already been mapped against the Human Genome.

$ time perl kraken2 --db DB30August --threads 4 --paired --classified-out cseqs#.fq --unclassified-out uncseqs#.fq ./Sequences/Pool1.out.mate1.fastq ./Sequences/Pool1.out.mate2.fastq

Loading database information... done.
0 sequences (0.00 Mbp) processed in 0.001s (0.0 Kseq/m, 0.00 Mbp/m).
0 sequences classified (-nan%)
0 sequences unclassified (-nan%)

real 7m55,107s
user 0m0,168s
sys 1m41,420s

The steps I have followed are:

Download taxonomy:
perl kraken2-build --download-taxonomy --db DB30August

Download libraries:
perl kraken2-build --download-library bacteria --db DB30August
perl kraken2-build --download-library fungi --db DB30August
perl kraken2-build --download-library plant --db DB30August

Build Database:
perl kraken2-build --build --db DB30August
*This took around 7 hours

Classification:
time perl kraken2 --db DB30August --threads 4 --paired --classified-out cseqs#.fq --unclassified-out uncseqs#.fq ./Sequences/Pool1.out.mate1.fastq ./Sequences/Pool1.out.mate2.fastq`

Thank you in advanced for your help :)

[adec37]

I am also having this problem.. has anyone found a fix?

[suburbianmantia]

I had the same kind of issue when the input .fastq file was not correctly written (due to typo or something similar). I would suggest to try rerunning the command inside the folder where your fastq files are. Just provide your fastq files full name and check that there is no typo and that the actual fastq files that you provide in your kraken2 command are accessible (for example : more your_file.fastq). Here you have 0 processed sequences which means in my opinion that kraken2 is not even able to access the sequences in your file.

[nicolo-tellini]

Hello,

I am getting the same output

Loading database information... done. 0 sequences (0.00 Mbp) processed in 6.356s (0.0 Kseq/m, 0.00 Mbp/m). 0 sequences classified (-nan%) 0 sequences unclassified (-nan%)

although I actually have non empty files with the reads classified. I am using kraken2 version 2.0.9-beta.

Is it maybe a bug ?

nic