# Having issues with Strainphlan **URL:** \ **Category:** StrainPhlAn\ **Created:** [February 19, 2020, 12:18am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218 "2020-02-19T00:18:56Z")\ **Posts on this page:** 16\ **Page:** 1
**Author:** ![mradz19](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/mradz19/32/112_2.png) [@mradz19](https://forum.biobakery.org/u/mradz19)\ **Post date:** [February 19, 2020, 12:18am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/1 "2020-02-19T00:18:56Z")
I downloaded metaphlan2 using conda and I am attempting to use strainphlan to profile my samples at the strain level. I have been following the instructions on this page: [https://bitbucket.org/biobakery/biobakery/wiki/strainphlan](https://bitbucket.org/biobakery/biobakery/wiki/strainphlan) I successfully completed the sample2markers.py step, however I cannot run step 3: Identify clades detected in the samples and build reference databases. The command i am using is: strainphlan.py --ifn\_samples p100\_bowtie2\_aligned.markers --output\_dir markers/ --print\_clades\_only \> clades.txt Which results in the following error: Traceback (most recent call last): File “/mnt/nfs/home/30041036/.conda/envs/metaphlan2/bin/strainphlan.py”, line 1585, in strainphlan() File “/mnt/nfs/home/30041036/.conda/envs/metaphlan2/bin/strainphlan.py”, line 1581, in strainphlan strainer(args) File “/mnt/nfs/home/30041036/.conda/envs/metaphlan2/bin/strainphlan.py”, line 1365, in strainer db = pickle.load(bz2.BZ2File(args[‘mpa\_pkl’])) File “/mnt/nfs/home/30041036/.conda/envs/metaphlan2/lib/python3.7/bz2.py”, line 92, in **init** self.\_fp = \_builtin\_open(filename, mode) IsADirectoryError: [Errno 21] Is a directory: ‘/mnt/nfs/home/30041036/.conda/envs/metaphlan2/bin/metaphlan\_databases’ How can I fix this? I can’t see that anyone else has had similar issues. Also in step 4 --ifn\_markers s\_\_Eubacterium\_siraeum.markers.fasta is used in the command, how do I generate this fasta file for the species I am interested in (e.g. staphylococcus aureus)? ---
**Author:** ![aitor.blancomiguez](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/aitor.blancomiguez/32/86_2.png) [@aitor.blancomiguez](https://forum.biobakery.org/u/aitor.blancomiguez)\ **Post date:** [February 19, 2020, 8:48am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/2 "2020-02-19T08:48:48Z")
Hi mradz19, Could you tell me the version of the MetaPhlAn2 database you used for the profiling? For the question about the step 4, you should use the script _extract\_markers.py_ You can take a look on this example (step 4): [https://bitbucket.org/biobakery/metaphlan2/src/default/README.md#markdown-header-usage](https://bitbucket.org/biobakery/metaphlan2/src/default/README.md#markdown-header-usage) For this script, remember to specify the correct metaphlan2 database version. Best, Aitor ---
**Author:** ![mradz19](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/mradz19/32/112_2.png) [@mradz19](https://forum.biobakery.org/u/mradz19)\ **Post date:** [February 19, 2020, 10:01pm UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/3 "2020-02-19T22:01:42Z")
Hi Aitor, This is the version: MetaPhlAn version 2.96.1 (02 Feb 2020) ---
**Author:** ![aitor.blancomiguez](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/aitor.blancomiguez/32/86_2.png) [@aitor.blancomiguez](https://forum.biobakery.org/u/aitor.blancomiguez)\ **Post date:** [February 20, 2020, 9:58am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/4 "2020-02-20T09:58:50Z")
Hi mradz19, Try to add the param _–index v296\_CHOCOPhlAn\_201901_ to the strainphlan execution like: strainphlan.py --ifn\_samples p100\_bowtie2\_aligned.markers --output\_dir markers/ --print\_clades\_only --index v296\_CHOCOPhlAn\_201901 \> clades.txt Best, Aitor ---
**Author:** ![mradz19](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/mradz19/32/112_2.png) [@mradz19](https://forum.biobakery.org/u/mradz19)\ **Post date:** [February 21, 2020, 2:35am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/5 "2020-02-21T02:35:47Z")
Hi Aitor, I tried adding that parameter and got the same error message. ---
**Author:** ![aitor.blancomiguez](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/aitor.blancomiguez/32/86_2.png) [@aitor.blancomiguez](https://forum.biobakery.org/u/aitor.blancomiguez)\ **Post date:** [February 21, 2020, 10:18am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/6 "2020-02-21T10:18:25Z")
> [@mradz19](#): > > /mnt/nfs/home/30041036/.conda/envs/metaphlan2/bin/metaphlan\_databases Hi Michael, Could you check then the content of this folder: /mnt/nfs/home/30041036/.conda/envs/metaphlan2/bin/metaphlan\_databases ---
**Author:** ![mradz19](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/mradz19/32/112_2.png) [@mradz19](https://forum.biobakery.org/u/mradz19)\ **Post date:** [February 23, 2020, 11:16pm UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/7 "2020-02-23T23:16:54Z")
Hi Aitor, These are the contents of that folder: mpa\_latest mpa\_v295\_CHOCOPhlAn\_201901.1.bt2 mpa\_v295\_CHOCOPhlAn\_201901.2.bt2 mpa\_v295\_CHOCOPhlAn\_201901.3.bt2 mpa\_v295\_CHOCOPhlAn\_201901.4.bt2 mpa\_v295\_CHOCOPhlAn\_201901.fna.bz2 mpa\_v295\_CHOCOPhlAn\_201901.md mpa\_v295\_CHOCOPhlAn\_201901.pkl mpa\_v295\_CHOCOPhlAn\_201901.rev.1.bt2 mpa\_v295\_CHOCOPhlAn\_201901.rev.2.bt2 mpa\_v295\_CHOCOPhlAn\_201901.tar I chaged the --index tag to v295\_CHOCOPhlan\_201901 and the command ran, however the output .txt file is empty. This is the log of the run: strainphlan.py --ifn\_samples p100\_bowtie2\_aligned.markers --output\_dir markers/ --print\_clades\_only --index v295\_CHOCOPhlAn\_201901 \> clades2.txt 2020-02-24 10:19:18,264 | INFO | **main** | strainer | 1364 | Load mpa\_pkl 2020-02-24 10:19:30,288 | INFO | **main** | strainer | 1380 | Get clades from db 2020-02-24 10:19:31,878 | INFO | **main** | strainer | 1425 | Get clades from samples 2020-02-24 10:19:31,878 | DEBUG | **main** | load\_sample | 1150 | load p100\_bowtie2\_aligned.markers ---
**Author:** ![CK\_zhu](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/ck_zhu/32/151_2.png) [@CK\_zhu](https://forum.biobakery.org/u/CK_zhu)\ **Post date:** [March 14, 2020, 4:00am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/8 "2020-03-14T04:00:04Z")
Hello I have the same problem, nothing output when I run the last step `strainphlan.py` ```auto #MetaPhlAn version 2.96.1 (02 Feb 2020) # run metaphlan2 on the demo sample input files >metaphlan2.py $INPUT_FOLDER/13530241_SF05.fasta.gz $OUTPUT_FOLDER/13530241_SF05_profile.txt --bowtie2out $OUTPUT_FOLDER/13530241_SF05_bowtie2.txt --samout $OUTPUT_FOLDER/13530241_SF05.sam.bz2 --input_type multifasta --index mpa_v296_CHOCOPhlAn_201901 --nproc $THREADS Elapsed time to run MetaPhlAn2: 61.940003871917725 s # run sample to markers on all of the samples >sample2markers.py --ifn_samples $OUTPUT_FOLDER/13530241_SF05.sam.bz2 --input_type sam --output_dir $OUTPUT_FOLDER --nprocs $THREADS /software/StrainPhlAn2/biobakery-biobakery-414eab928577/demos/biobakery_demos/data/strainphlan/output/13530241_SF05.sam.bz2 | samtools view -bS - | samtools sort - -o /software/StrainPhlAn2/biobakery-biobakery-414eab928577/demos/biobakery_demos/data/strainphlan/output/13530241_SF05.sam.bz2.bam.sorted | samtools mpileup -u - | bcftools view -c -g -p 1.1 - | fix_AF1.py --input_file - | vcfutils.pl vcf2fq # run metaphlan2 strainer on all samples (add the flag to reduce the default as these are subsampled) strainphlan.py --index v296_CHOCOPhlAn_201901 --ifn_samples $OUTPUT_FOLDER/*.markers --ifn_markers $INPUT_FOLDER/s __Eubacterium_siraeum.markers.fasta --ifn_ref_genomes $INPUT_FOLDER/GCF_000154325.fna.bz2 --output_dir $OUTPUT_FOLDER --nprocs_main $THREADS --clades s__ Eubacterium_siraeum --marker_in_clade 0.2 --keep_alignment_files 2020-03-14 11:40:15,504 | INFO | __main__ | strainer | 1364 | Load mpa_pkl 2020-03-14 11:40:25,664 | INFO | __main__ | strainer | 1380 | Get clades from db 2020-03-14 11:40:28,017 | INFO | __main__ | strainer | 1444 | Add reference genomes 2020-03-14 11:40:28,032 | DEBUG | __main__ | add_ref_genomes | 617 | add 1 reference genomes ... 2020-03-14 11:40:28,495 | DEBUG | __main__ | filter_sequence | 475 | sample GCF_000154325, number of markers after N_in_marker: 150 sample GCF_000154325, number of markers after marker_strip_length: 150 2020-03-14 11:40:28,495 | DEBUG | __main__ | strainer | 1503 | remove samples with percentage of markers less than marker_in_clade 2020-03-14 11:40:28,495 | DEBUG | __main__ | build_tree | 850 | skip clade s__Eubacterium_siraeum because number of present samples is 1 2020-03-14 11:40:28,495 | INFO | __main__ | strainer | 1550 | Finished! ``` ---
**Author:** ![aitor.blancomiguez](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/aitor.blancomiguez/32/86_2.png) [@aitor.blancomiguez](https://forum.biobakery.org/u/aitor.blancomiguez)\ **Post date:** [March 16, 2020, 8:42am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/9 "2020-03-16T08:42:14Z")
Hi Michael, When StrainPhlAn is not able to return any clade could be due two main reasons: 1. The database version you used for create the SAM file is different than the version you used for executing StrainPhlAn. This can be checked taking a look on the first line of the abundances report file generated together with the SAM file. 2. The sample2markers script was not able to reconstruct enough markers for your sample. If you could share your markers file I could take a deeper look on the problem. Best, Aitor ---
**Author:** ![aitor.blancomiguez](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/aitor.blancomiguez/32/86_2.png) [@aitor.blancomiguez](https://forum.biobakery.org/u/aitor.blancomiguez)\ **Post date:** [March 16, 2020, 8:45am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/10 "2020-03-16T08:45:30Z")
Hi CK\_zhu, As you can see in the lines returned by StrainPhlAn: > 2020-03-14 11:40:28,495 | DEBUG | **main** | build\_tree | 850 | skip clade s\_\_Eubacterium\_siraeum because number of present samples is 1 StrainPhlAn only detected the clade in one of your files, so the strain-level analysis is imposible to execute. Best, Aitor ---
**Author:** ![lzh1982](https://avatars.discourse-cdn.com/v4/letter/l/5fc32e/32.png) [@lzh1982](https://forum.biobakery.org/u/lzh1982)\ **Post date:** [November 20, 2020, 2:18am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/11 "2020-11-20T02:18:37Z")
Hi Aitor, Where I can download the STRAINPHLAN\_DB\_REFERENCE and STRAINPHLAN\_DB\_MAKERS directly?Thank you very much! Best regards Li Zhihua ---
**Author:** ![aitor.blancomiguez](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/aitor.blancomiguez/32/86_2.png) [@aitor.blancomiguez](https://forum.biobakery.org/u/aitor.blancomiguez)\ **Post date:** [November 20, 2020, 10:31am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/12 "2020-11-20T10:31:40Z")
Hi @lzh1982 The StrainPhlAn markers’ database is same as the MetaPhlAn markers’ database. If you installed MetaPhlAn 3 via conda, StrainPhlAn and the markers’ database will be also downloaded and installed, please check the tutorial for more info: [https://github.com/biobakery/MetaPhlAn/wiki/MetaPhlAn-3.0#installation](https://github.com/biobakery/MetaPhlAn/wiki/MetaPhlAn-3.0#installation) If you have any issue with the conda installatioiin, you can also download the database from the following links: - [Dropbox](https://www.dropbox.com/sh/7qze7m7g9fe2xjg/AADHWzATSQcI0CNFD0sk7MAga) - [Google Drive](https://drive.google.com/drive/folders/1_HaY16mT7mZ_Z8JtesH8zCfG9ikWcLXG?usp=sharing) - [Zenodo](https://zenodo.org/record/3957592) Best, Aitor ---
**Author:** ![lzh1982](https://avatars.discourse-cdn.com/v4/letter/l/5fc32e/32.png) [@lzh1982](https://forum.biobakery.org/u/lzh1982)\ **Post date:** [November 21, 2020, 3:13am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/13 "2020-11-21T03:13:16Z")
Hi Aitor, Thank you very much for your explanation! I have installed biobakery\_workflow through docker. I need know how to download the reference database not only maker database and install STRAINPHLAN\_DB\_REFERENCE manually? Would you help me? Best regards Li Zhihua ---
**Author:** ![fbeghini](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fbeghini/32/81_2.png) [@fbeghini](https://forum.biobakery.org/u/fbeghini)\ **Post date:** [November 23, 2020, 2:51pm UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/14 "2020-11-23T14:51:15Z")
Hi, You should set the the environmental variables STRAINPHLAN\_DB\_REFERENCE and STRAINPHLAN\_DB\_MAKERS inside your Docker instance using `export`. `STRAINPHLAN_DB_REFERENCE` should point to the folder containing the reference genomes used when running StrainPhlAn and `STRAINPHLAN_DB_MARKERS` points to the folder containing the StrainPhlAn marker files. ---
**Author:** ![lzh1982](https://avatars.discourse-cdn.com/v4/letter/l/5fc32e/32.png) [@lzh1982](https://forum.biobakery.org/u/lzh1982)\ **Post date:** [November 24, 2020, 11:53pm UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/15 "2020-11-24T23:53:37Z")
Dear Dr.Francesco.Beghini, Thank you very much for your explanation! I do not know where I can download the `STRAINPHLAN_DB_REFERENCE` database. Because I can not install directly using the order:`biobakery_workflows --install wmgx, so I want to download the corresponding datatbase and install manually! Many thanks!` `Best regards` `Li Zhihua` ---
**Author:** ![fbeghini](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/fbeghini/32/81_2.png) [@fbeghini](https://forum.biobakery.org/u/fbeghini)\ **Post date:** [November 27, 2020, 10:26am UTC](https://forum.biobakery.org/t/having-issues-with-strainphlan/218/16 "2020-11-27T10:26:47Z")
You should retrieve the genomes of the species of interest from any genomic repository (e.g. Refseq)