Abundant phylum not being classified

[Manso002]

Hello all. I have run a squeeze meta project in sequential mode (my workstation can't run other modes with my samples) and the results show that in every single one of my 12 samples the top 2-3 most abundant taxa is represented as 'Unclassified bacteria'. I tried running the samples with several mapping and assembly software but i keep getting the same results. I was wondering if you knew a way i can assign taxonomy to it or if my samples are contaminated. Can't think of a solution. Thanks in advance.

[jtamames]

Hello Are you having the same results using sqm_reads or sqm_longreads? These are not relying in the assembly, just to be sure the problem is not there. I also recommend you: locate some of these unclassified ORFs. Check the Diamond results for these (04 file in intermediate directory). Then run a blastp search of the same ORF in NCBI, and compare results. And let me know :)

Best, J

[fpusan]

Also is this by any chance a metatranscriptome?

[Manso002]

Thanks for the fast response! I didn't try sqm_longreads. I will try both approaches u suggest me and let you know.

@fpusan I actually have metagenome samples

[Manso002]

Hello again. I am trying to run sqm_longreads and i get the error:

Error running /home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/bin/diamond blastx -q /home/meridian/ANE1/ANETO_1/ANETO_1_R1.fastq -p 12 -d /home/meridian/miniconda3/envs/SqueezeMeta2/db/nr.dmnd -f tab -F 15 --quiet --range-culling -b 15.4 -e 0.001 --id 30 --top 10 -o /home/meridian/ANE1/ANETO_1/ANETO_1_R1.fastq.gz.nr.blastx -f 6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore qlen slen at /home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/utils/sqm_longreads.pl line 933.

So i tried sqm_reads to see if i get the same error with blasx and that happens too. I can't figure out how to fix it. Also, I am not sure how to search for unclassified ORFS in Diamond results. The files being that huge makes it difficult to find unclassified orfs. I was wondering if you could help me.

Thanks in advance

[jtamames]

Hello What happens if you do /home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/bin/diamond If that works, try giving the full command and check the possible error message:

/home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/bin/diamond blastx -q /home/meridian/ANE1/ANETO_1/ANETO_1_R1.fastq -p 12 -d /home/meridian/miniconda3/envs/SqueezeMeta2/db/nr.dmnd -f tab -F 15 --quiet --range-culling -b 15.4 -e 0.001 --id 30 --top 10 -o /home/meridian/ANE1/ANETO_1/ANETO_1_R1.fastq.gz.nr.blastx -f 6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore qlen slen

For selecting the ORFs, just take a look at the 06.wranks file in the results directory. Locate some unclassified ORFs, and then get the aa sequence from the 03.faa file also in the results directory Best,

[Manso002]

I get this error when running the command you suggested:

Error: Syntax: diamond COMMAND [OPTIONS]. To print help message: diamond help.

When calling for diamond, i get an error as if it is not installed. This is weird since diamond runs smoothly when running squeezemeta.pl.

[jtamames]

But /home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/bin/diamond returns anything?

[Manso002]

Hi. It returns the error I commented you: Error: Syntax: diamond COMMAND [OPTIONS]. To print help message: diamond help.

Test_install.pl tells me everything is ok

[jtamames]

it does look ok, otherwise the error message would be diamond: not found, or similar. And then what is the output of /home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/bin/diamond blastx?

[Manso002]

I get this: Error: Missing parameter: database file (--db/-d).

Thanks for the patience Javier, much appreciated.

[jtamames]

Then it is working properly. And now, what if you do /home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/bin/diamond blastx -q /home/meridian/ANE1/ANETO_1/ANETO_1_R1.fastq -p 12 -d /home/meridian/miniconda3/envs/SqueezeMeta2/db/nr.dmnd -f tab -F 15 --quiet --range-culling -b 15.4 -e 0.001 --id 30 --top 10 -o /home/meridian/ANE1/ANETO_1/ANETO_1_R1.fastq.gz.nr.blastx?

[Manso002]

I get this: No such file or directory Error: Error calling stat on file /home/meridian/ANE1/ANETO_1/ANETO_1_R1.fastq

[jtamames]

Ok... could you check your samples file, to see if the fastq files you specify there are placed in the proper locations?

[Manso002]

Files are correctly located, yes. Could the error be related to a memory issue? I am running some other resource-consumming processes and came to my mind that could be the problem.

[jtamames]

No, the error is that diamond is not finding the file /home/meridian/ANE1/ANETO_1/ANETO_1_R1.fastq. Could it be that it is gunzipped and therefore the name does not match?

[Manso002]

fastq files are gunzipped, you are right. On the other hand, there are no fastq files of any kind in /home/meridian/ANE1/ANETO_1/.

[jtamames]

I am asking for that path because it is the one taht apparently you provided when running the script. Please check everything carefully (files, filenames, directories, etc) and try again.

[Manso002]

I will try again double checking everything and let you know. Once again, thank you Javier.

[Manso002]

Hello all. Sorry to bother you again. I have been trying to run sqm_reads and sqm_long reads following your recommendations @jtamames and it is not working. The code i used is this:

/home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/utils/sqm_reads.pl -p ANE1 -s /home/meridian/ANETO/sqm_reads_aneto1.txt -f /home/meridian/ANETO/ANETO_reads

But i again get the following error:

[59 seconds]: Running Diamond (Buchfink et al 2015, Nat Methods 12, 59-60) for taxa (GenBank nr, Clark et al 2016, Nucleic Acids Res 44, D67-D72) Error running command /home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/bin/diamond blastx -q /home/meridian/ANETO/ANETO_reads/ANETO_1_R1.fastq.gz -p 12 -d /home/meridian/miniconda3/envs/SqueezeMeta2/db/nr.dmnd -e 0.001 --quiet -f tab -b 15.2 -o ANE1/ANETO_1/ANETO_1_R1.fastq.gz.tax.m8 at /home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/utils/sqm_reads.pl line 245.

Hope you can help me. Thanks and have a nice day!

[jtamames]

Have you checked if your samples are in /home/meridian/ANETO/ANETO_reads?

[Manso002]

Yes, samples are in that path.

[jtamames]

Ok, then what is the result of running: /home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/bin/diamond blastx -q /home/meridian/ANETO/ANETO_reads/ANETO_1_R1.fastq.gz -p 12 -d /home/meridian/miniconda3/envs/SqueezeMeta2/db/nr.dmnd o ANE1/ANETO_1/ANETO_1_R1.fastq.gz.tax.m8

[Manso002]

Error: Invalid parameter count for option '--db'

[jtamames]

Sorry, mistake. Run it again: /home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/bin/diamond blastx -q /home/meridian/ANETO/ANETO_reads/ANETO_1_R1.fastq.gz -p 12 -d /home/meridian/miniconda3/envs/SqueezeMeta2/db/nr.dmnd -o ANE1/ANETO_1/ANETO_1_R1.fastq.gz.tax.m8

[Manso002]

It is running. Although ANETO_1_R1.fastq.gz.tax.m8 has no info in it (0 bytes). I assume that should not happen right?

diamond v2.0.15.153 (C) Max Planck Society for the Advancement of Science Documentation, support and updates available at http://www.diamondsearch.org Please cite: http://dx.doi.org/10.1038/s41592-021-01101-x Nature Methods (2021)

CPU threads: 12

Scoring parameters: (Matrix=BLOSUM62 Lambda=0.267 K=0.041 Penalties=11/1) Temporary directory: ANE1/ANETO_1

Target sequences to report alignments for: 25

Opening the database... [0.08s] Database: /home/meridian/miniconda3/envs/SqueezeMeta2/db/nr.dmnd (type: Diamond database, sequences: 595907626, letters: 234169316349) Block size = 2000000000

[jtamames]

Okl. Please stop it and run the full command: /home/meridian/miniconda3/envs/SqueezeMeta2/SqueezeMeta/bin/diamond blastx -q /home/meridian/ANETO/ANETO_reads/ANETO_1_R1.fastq.gz -p 12 -d /home/meridian/miniconda3/envs/SqueezeMeta2/db/nr.dmnd -e 0.001 --quiet -f tab -b 15.2 -o ANE1/ANETO_1/ANETO_1_R1.fastq.gz.tax.m8

[Manso002]

It seems like its running, although no output messages are shown like before. I will let you know if anything goes wrong.

Thanks a lot Javier

[fpusan]

Closing due to lack of activty, feel free to reopen