ERROR: Exception: [Errno 2] No such file or directory: '/tmp/atram_bku5sgqt/lib_des-1S_uce-5k-probes.fasta_01_owavb3if/trinity.Trinity.fasta'

[liugang9988]

Hi,

I am doing the aTRAM, but after running the atram.py, it reported the error like below, could you please help take a loot what is going on? Thank you very much

2020-11-16 02:16:33 INFO : ################################################################################
2020-11-16 02:16:33 INFO : aTRAM version: v2.3.4
2020-11-16 02:16:33 INFO : Python version: 3.6.3 |Anaconda, Inc.| (default, Oct 13 2017, 12:02:49) [GCC 7.2.0]
2020-11-16 02:16:33 INFO : aTRAM/atram.py --blast-db=/lib/lib_des-1S --query=UCE_sub/uce-5k-probes.fasta --assembler=trinity --output-prefix=des-1S/output_nucleotide/des-1S --log-file des-1S/Test.log -i 5 --cpus 10
2020-11-16 02:16:33 INFO : aTRAM blast DB = "/lib/lib_des-1S", query = "uce-5k-probes.fasta", iteration 1
2020-11-16 02:16:33 INFO : Blasting query against shards: iteration 1
2020-11-16 02:16:44 INFO : All 9 blast results completed
2020-11-16 02:16:44 INFO : 1 blast hits in iteration 1
2020-11-16 02:16:44 INFO : Writing assembler input files: iteration 1
2020-11-16 02:16:44 INFO : Assembling shards with trinity: iteration 1
2020-11-16 02:16:45 ERROR: Exception: Command 'Trinity --seqType fa --max_memory 38G --CPU 10 --output '/tmp/atram_bku5sgqt/lib_des-1S_uce-5k-probes.fasta_01_owavb3if/trinity' --full_cleanup --no_bowtie --left '/tmp/atram_bku5sgqt/lib_des-1S_uce-5k-probes.fasta_01_owavb3if/paired_1.fasta' --right '/tmp/atram_bku5sgqt/lib_des-1S_uce-5k-probes.fasta_01_owavb3if/paired_2.fasta'' returned non-zero exit status 1.
2020-11-16 02:16:45 ERROR: Exception: [Errno 2] No such file or directory: '/tmp/atram_bku5sgqt/lib_des-1S_uce-5k-probes.fasta_01_owavb3if/trinity.Trinity.fasta'

[juliema]

it looks like it is having a hard time finding the temp file. One way to get around this would be to go ahead and set the temp file yourself. so add this to the command

…

--temp-dir <path to an existing temp file you want the files to go into>

On Mon, Nov 16, 2020 at 2:56 AM liugang9988 ***@***.***> wrote: Hi, I am doing the aTRAM, but after running the atram.py, it reported the error like below, could you please help take a loot what is going on? Thank you very much 2020-11-16 02:16:33 INFO : ################################################################################ 2020-11-16 02:16:33 INFO : aTRAM version: v2.3.4 2020-11-16 02:16:33 INFO : Python version: 3.6.3 |Anaconda, Inc.| (default, Oct 13 2017, 12:02:49) [GCC 7.2.0] 2020-11-16 02:16:33 INFO : aTRAM/atram.py --blast-db=/lib/lib_des-1S --query=UCE_sub/uce-5k-probes.fasta --assembler=trinity --output-prefix=des-1S/output_nucleotide/des-1S --log-file des-1S/Test.log -i 5 --cpus 10 2020-11-16 02:16:33 INFO : aTRAM blast DB = "/lib/lib_des-1S", query = "uce-5k-probes.fasta", iteration 1 2020-11-16 02:16:33 INFO : Blasting query against shards: iteration 1 2020-11-16 02:16:44 INFO : All 9 blast results completed 2020-11-16 02:16:44 INFO : 1 blast hits in iteration 1 2020-11-16 02:16:44 INFO : Writing assembler input files: iteration 1 2020-11-16 02:16:44 INFO : Assembling shards with trinity: iteration 1 2020-11-16 02:16:45 ERROR: Exception: Command 'Trinity --seqType fa --max_memory 38G --CPU 10 --output '/tmp/atram_bku5sgqt/lib_des-1S_uce-5k-probes.fasta_01_owavb3if/trinity' --full_cleanup --no_bowtie --left '/tmp/atram_bku5sgqt/lib_des-1S_uce-5k-probes.fasta_01_owavb3if/paired_1.fasta' --right '/tmp/atram_bku5sgqt/lib_des-1S_uce-5k-probes.fasta_01_owavb3if/paired_2.fasta'' returned non-zero exit status 1. 2020-11-16 02:16:45 ERROR: Exception: [Errno 2] No such file or directory: '/tmp/atram_bku5sgqt/lib_des-1S_uce-5k-probes.fasta_01_owavb3if/trinity.Trinity.fasta' — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#298>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AA2AZ3XGFTLPYUXLQENY2Q3SQDLL5ANCNFSM4TW2MUDA> .

[liugang9988]

I tried adding --temp-dir, but the same errors were reported, although spade, velvet were used. Could you explain a little bit more about the debugging issue posted here?, https://github.com/juliema/aTRAM/blob/master/doc/troubleshooting.md#Debugging-assembler-issues

I tried to follow the steps, but it seemed nothing was generated in the output directory. This thing bothered me a long time. Thanks.

[mylena-s]

Hi everyone,
I had the same issue running aTRAM in a server with PBS queues. I found that atram was executing a newer version of Trinity that was already installed in the server. So I solved it by exporting the path where the other tirnity version was installed. Maybe this info might help you to ressolve the issue.

Cheers

[rafelafrance]

One thing to try is the --path option. This prepends a directory so it can find the correct version of a file. I have installed the assemblers in odd places (like my private bin directory) so I'll do something like --path /home/rafe/bin

[SidG13]

Hi everyone,

I had the same issue, atram.py has issues finding temporary files. I only have a single installation of velvetg/velveth and Trinity which I installed via conda and so are in ~/miniconda3/bin/. I used both --path pointing to this path and --temp-dir as suggested above after creating a directory, but neither have worked. I'm running aTRAM v2.4.3 and python v3.8.5

In case it helps, here's my system information (output of uname -a):
Linux sysname 3.13.0-170-generic #220-Ubuntu SMP Thu May 9 12:40:49 UTC 2019 x86_64 x86_64 x86_64 GNU/Linux

Any other suggestions? Thanks very much!

[rafelafrance]

@SidG13 Your actions to fix this seem on target but the setup is not something I've personally seen before. Could you provide me with the specific error you're getting and the atram parameters you're using? I'll look into this.

[zgliwen]

@rafelafrance
hi,

I also had the same issue. The difference from others was that I used Abyss rather than Trinity as the assembler, and it reported the similar error:
ERROR: Exception: [Errno 2] No such file or directory: '/data/results/atram_out_220407/kaM_49223_imd/atram_qfz_iu_n/kaM_49223_seq.fa_01_xdlazpp6/output.fasta-unitigs.fa'

My command was:
$ atram.py --database atram_out_220407/kaM_49223 --query basic_info/kaM_seq.fa --assembler abyss -t /data/results/atram_out_220407/kaM_49223_imd --keep-temp-dir --no-filter --path miniconda3/envs/py3/bin -o /data/results/atram_out_220407/kaM_49223

However, with the fasta files created by atram in the temporary directory, I can get the assemble by Abyss-pe, and the command was
$ abyss-pe np=8 name=/data/results/atram_out_220407/kaM_49223_abyss k=64 in='atram_out_220407/kaM_49223_imd/atram_qfz_iu_n/kaM_49223_seq.fa_01_xdlazpp6/paired_1.fasta atram_out_220407/kaM_49223_imd/atram_qfz_iu_n/kaM_49223_seq.fa_01_xdlazpp6/paired_2.fasta'

The version of softwares I used were:
aTRAM 2.4.3
abyss 2.3.4-h41cdee2_1

Could you give me some suggestions, please? Thanks for your help.

[rafelafrance]

@zgliwen I know they may look the same but this is actually a different issue. I'll look into it ASAP.

Let's move this discussion over to issue #308

[zgliwen]

@zgliwen I know they may look the same but this is actually a different issue. I'll look into it ASAP.

Let's move this discussion over to issue #308

Okay, thanks. Sorry that I couldn't tell the differences. And looking forward to your replay.

[NikoHensley]

Hi! I'm really interested in this tool, so thank you for developing it. I've been playing around with the program and have received the same error after a few successful runs.

I made a database with a set of paired end reads from Illumina HiSeq data. During the aTRAM step, I used a protein query to successfully blast, assemble, and iterate through to contig output, as below:

Call:
./atram.py -b ../atram_test_egd/egd -Q ../luc_bait/luc_bait_pro.fasta -o ../atram_egd/egd -a trinity -i 10

However, when I tried to use aTRAM with a DNA query (the full mRNA from the translated proteins, introns included), then the program gave me this error, indicating it could not find (presumably create) the temporary file:

Call:
./atram.py -b ../atram_test_egd/egd -Q ../luc_bait/luc_bait_full_mRNA_hilg_noc.txt -o ../atram_egd/egd -a trinity -i 10

Error:

2023-11-21 13:17:16.680950 INFO : ################################################################################
(    9910) 2023-11-21 13:17:16.681060 INFO : aTRAM version: v2.4.4
(    9910) 2023-11-21 13:17:16.681091 INFO : Python version: 3.11.5 (main, Sep 11 2023, 13:23:44) [GCC 11.2.0]
(    9910) 2023-11-21 13:17:16.681118 INFO : ./atram.py -b ../atram_test_egd/egd -Q ../luc_bait/luc_bait_full_mRNA_hilg_noc.txt -o ../atram_egd/egd -a trinity -i 10
(    9910) 2023-11-21 13:17:16.681174 INFO : aTRAM blast DB = "../atram_test_egd/egd", query = "luc_bait_full_mRNA_hilg_noc_AB159608_1_1.fasta", iteration 1
(    9910) 2023-11-21 13:17:16.681346 INFO : Blasting query against shards: iteration 1
(    9910) 2023-11-21 13:17:23.228339 INFO : All 28 blast results completed
(    9910) 2023-11-21 13:17:23.228774 INFO : 3 blast hits in iteration 1
(    9910) 2023-11-21 13:17:23.228817 INFO : Writing assembler input files: iteration 1
(    9910) 2023-11-21 13:17:23.323726 INFO : Assembling shards with trinity: iteration 1
(    9910) 2023-11-21 13:18:26.974465 ERROR: Exception: [Errno 2] No such file or directory: '/tmp/atram__hl9tfj2/egd_luc_bait_full_mRNA_hilg_noc_AB159608_1_1.fasta_01_37drihy7/trinity.Trinity.fasta'

I have tried used the -t DIR flag to indicate a temporary directory, which gets created successfully but then seemingly the assembly itself within the temporary directory does not because the error above persists. When I examine the contents of the temp folder (attached image), I can see that the trinity folder is created, and the program is initialized but there is no final "/trinity.Trinity.fasta" output.

Interestingly, the blast results are MUCH smaller with the mRNA queries (3 hits) than with the translated protein queries (200,000+ hits). I thought then that maybe assembly is failing because of the hits (low number). Even when I add a --no-filter flag to remove the bit score stringency, the blast fails to find any more reads within the database that map to my mRNA queries.

I am not sure if assembly failure is due to low database matching rates with other people, but I thought I'd post this semi-deep dive into the issue. It doesn't seem intuitive to me that the read matching should be that low to the mRNA query compared to the translated proteins, but I think that's the main issue I'm experiencing.

I am not too familiar with these types of tools, but I wonder if using a read mapper or a Magic-BLAST type of tool would help more than traditional BLAST with my specific issue (i.e. pulling more reads to iteratively assemble given an mRNA query)?

I'm using a conda environment with python 3.11.5 and aTRAM 2.4.4