Something Error of KMA

[TaoCheng98]

Hi！ When I was ready to use ccMetagen, I found that I had some problems. At first, after configuring the database, I used rush to parallel kma. It didn't give an error, but it didn't generate any data either. The result files were empty. I then tried to abandon parallelism, but CCMetagen didn't work because of the "Uneven number of paired end files". I tried to detect my sequence file. In fact, R1 and R2 share the same number of reads. So I don't understand this error.（This error is shown in my attachment） I finally tried single-ended mapping and got the same result as before, no error but no data either.（This error is shown in my attachment） It looks like I didn't even get the first step, which is confusing. I can't solve the problem I'm having, but I do want to use CCMetagen, and if you can help me, I'd really appreciate it.

`(ccmetagen) t010208@bio3-Horsea-12U:~/Chengtao/meta/yuanlab2$ seqkit stat temp/qc/2017-HB-Ae-aF_1.fastq file format type num_seqs sum_len min_len avg_len max_len temp/qc/2017-HB-Ae-aF_1.fastq FASTQ DNA 10,002,631 1,239,361,408 31 123.9 150 (ccmetagen) t010208@bio3-Horsea-12U:~/Chengtao/meta/yuanlab2$ seqkit stat temp/qc/2017-HB-Ae-aF_2.fastq file format type num_seqs sum_len min_len avg_len max_len temp/qc/2017-HB-Ae-aF_2.fastq FASTQ DNA 10,002,631 1,221,789,497 31 122.1 150

(ccmetagen) t010208@bio3-Horsea-12U:~/Chengtao/meta/yuanlab2$ kma -ipe temp/qc/2018-GZ-Ae-p_1.fastq temp/qc/2018-GZ-Ae-p_2.fastq temp/kma/2018-GZ-Ae-p_2_kma -t_db /home/data/refdir/database/kraken2/CCMetagen/ncbi_nt_no_env_11jun2019 -t 40 -1t1 -mem_mode -and -apm f

Uneven number of paired end files.

I can not understand this error "Uneven number of paired end files".

(ccmetagen) t010208@bio3-Horsea-12U:~/Chengtao/meta/yuanlab2$ kma -i temp/qc/2018-GZ-Ae-p_1.fastq -o temp/kma/2018-GZ-Ae-p_kma -t_db /home/data/refdir/database/kraken2/CCMetagen/ncbi_nt_no_env_11jun2019 -t 30 -1t1 -mem_mode -and (ccmetagen) t010208@bio3-Horsea-12U:~/Chengtao/meta/yuanlab2$ ls -al ./temp/kma/ total 16 drwxrwxr-x 2 t010208 t010208 4096 10月 27 01:13 . drwxrwxr-x 4 t010208 t010208 4096 10月 26 23:18 .. -rw-rw-r-- 1 t010208 t010208 0 10月 27 01:13 2018-GZ-Ae-p_kma.aln -rw-rw-r-- 1 t010208 t010208 20 10月 27 01:22 2018-GZ-Ae-p_kma.frag.gz -rw-rw-r-- 1 t010208 t010208 0 10月 27 01:13 2018-GZ-Ae-p_kma.fsa -rw-rw-r-- 1 t010208 t010208 129 10月 27 01:22 2018-GZ-Ae-p_kma.res

No error was reported, but no data was generated`

ccmetagen.txt

[vrmarcelino]

Hi Tao,

I think you are missing the flag '-o' to indicate the output filepath in KMA. KMA is complaining about an 'Uneven number of files', not sequences in the files. So I believe KMA is thinking that your output filepath is an additional sequence file. I also think that 40 threads will not make it efficient. Try it with 4 to start:

kma -ipe temp/qc/2018-GZ-Ae-p_1.fastq temp/qc/2018-GZ-Ae-p_2.fastq -o temp/kma/2018-GZ-Ae-p_2_kma -t_db /home/data/refdir/database/kraken2/CCMetagen/ncbi_nt_no_env_11jun2019 -t 4 -1t1 -mem_mode -and -apm f

Let me know if it works.

[TaoCheng98]

Hi vrmarcelino！

I followed your instructions, but the problem here is just like I mentioned before. It didn't give an error, but it didn't generate any data either. The result files were empty.

`(ccmetagen) t010208@bio3-Horsea-12U:~/Chengtao/meta/yuanlab2$ kma -ipe temp/qc/2018-GZ-Ae-p_1.fastq temp/qc/2018-GZ-Ae-p_2.fastq -o temp/kma/2018-GZ-Ae-p_2_kma -t_db /home/data/refdir/database/kraken2/CCMetagen/ncbi_nt_no_env_11jun2019 -t 4 -1t1 -mem_mode -and -apm f Collecting k-mer scores. Reading inputfile: temp/qc/2018-GZ-Ae-p_1.fastq temp/qc/2018-GZ-Ae-p_2.fastq Phred scale: 33

Total time used for DB loading: 139.61 s.

Finding k-mer ankers

Total number of query fragment after trimming: 12820422

Query converted

Query ankered

Score collection done Sort, output and select k-mer alignments. Total time for sorting and outputting KMA alignment 0.18 s.

Doing local assemblies of found templates, and output results Total time used for local assembly: 91.29 s.

Closing files (ccmetagen) t010208@bio3-Horsea-12U:~/Chengtao/meta/yuanlab2$ ls -al ./temp/kma/ total 24 drwxrwxr-x 2 t010208 t010208 4096 10月 28 11:10 . drwxrwxr-x 5 t010208 t010208 4096 10月 27 08:44 .. -rw-rw-r-- 1 t010208 t010208 0 10月 28 11:10 2018-GZ-Ae-p_2_kma.aln -rw-rw-r-- 1 t010208 t010208 20 10月 28 11:17 2018-GZ-Ae-p_2_kma.frag.gz -rw-rw-r-- 1 t010208 t010208 0 10月 28 11:10 2018-GZ-Ae-p_2_kma.fsa -rw-rw-r-- 1 t010208 t010208 129 10月 28 11:17 2018-GZ-Ae-p_2_kma.res -rw-rw-r-- 1 t010208 t010208 0 10月 27 01:13 2018-GZ-Ae-p_kma.aln -rw-rw-r-- 1 t010208 t010208 20 10月 27 01:22 2018-GZ-Ae-p_kma.frag.gz -rw-rw-r-- 1 t010208 t010208 0 10月 27 01:13 2018-GZ-Ae-p_kma.fsa -rw-rw-r-- 1 t010208 t010208 129 10月 27 01:22 2018-GZ-Ae-p_kma.res `

[vrmarcelino]

That is strange. Are all KMA files empty?

Could you perhaps try with a different sample for us to identify where the issue is? (e.g. https://github.com/vrmarcelino/CCMetagen/blob/master/tutorial/figs_tutorial/Subset_bird_data.zip)

Could you also try updating KMA to the latest version?

It is also possible that there is simply no match between your samples and the database. I think it could happen if the sequencing depth is too shallow and you have very high diversity, or if the same is from a very different environment. What type of samples are you working with? Also - are those raw short-read data?

Thanks!

[TaoCheng98]

Hi vrmarcelino！ Sorry for the late reply. As you suggested, I used the test data to run the program.(e.g. https://github.com/vrmarcelino/CCMetagen/blob/master/tutorial/figs_tutorial/Subset_bird_data.zip) Unfortunately, I got the same result as before. No errors, no results. And, during that time, I made a friend who had the same problem. Instead of using conda, he followed the installation process as mentioned. And he and I both use the latest KMA, 1.4.7. So, I don't know where our problem is. If you can help us, we will be very grateful to you. ~/Soft/kma/kma -ipe Avocet_Outback.R1.fastq Avocet_Outback.R2.fastq -o A1_out_kma -t_db /home/data/refdir/database/kraken2/CCMetagen/ncbi_nt_no_env_11jun2019 -t 12 -1t1 -mem_mode -and -apm f

[vrmarcelino]

Hi! Thanks for this. I haven't come across this issue before. Could you please try using KMA version 1.3.13? It might be a compatibility issue.

Thanks!

[ptlcc]

Hi Tao

This looks like an error related to kma. I have tested it with Vanessas suggestion above with different versions of kma, and there is indeed a problem. I have solved this in version 1.4.8 of kma.

Best, Philip

[vrmarcelino]

Thanks Philip!!