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MaestSi / MetONTIIME

A Meta-barcoding pipeline for analysing ONT data in QIIME2 framework
GNU General Public License v3.0
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error in evaluating the argument 'x' #55

Closed ysuzuki1978 closed 1 year ago

ysuzuki1978 commented 1 year ago

I would very much like to use MetONTIME for long read sequences and to proceed with 16S analysis. However, when I run Launch_MinION_mobile_lab.sh, I get the following error and it stops.

In MinION_mobile_lab.R.

args <- commandArgs(trailingOnly=TRUE) config_file_ind <- grep(x = args, pattern = "\.R")

We think that the "config_file_ind" is not working, but what is the solution?

Here is the message after the run.

Welcome to MinION meta-barcoding mobile laboratory!

Raw reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5 Basecalled reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/basecalling Preprocessing directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/preprocessing Reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/analysis Flow-cell: FLO-MIN106 Kit: SQK-PBK004 PCR primers trimming length: 25 bp Barcodes used in this experiment: BC01, BC02, BC03, BC04, BC05, BC06, BC07, BC08, BC09, BC10, BC11, BC12 Basecalling is going to be performed by : Guppy Basecalling Software, (C) Oxford Nanopore Technologies plc. Version 6.4.2+97a7f06, minimap2 version 2.24-r1122 Basecalling model: default Demultiplexing is going to be performed by guppy_barcoder after basecalling

Error in h(simpleError(msg, call)) : error in evaluating the argument 'x' in selecting a method for function 'grep': cannot open the connection Calls: grep -> readLines -> file -> .handleSimpleError -> h In addition: Warning messages: 1: In file(con, "r") : 'raw = FALSE' but '/home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/analysis/' is not a regular file 2: In file(con, "r") : cannot open file '/home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/analysis/': it is a directory Execution halted

MaestSi commented 1 year ago

Hi, what is the exact command that you ran? I suspect there might have been an issue either during base-calling or demultiplexing. Can you show me the content of /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/basecalling, /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/preprocessing and /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/analysis folders? Thanks, Simone

ysuzuki1978 commented 1 year ago

Thank you for your comments.

/home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/analysis

-rw-rw-r-- 1 yakuri yakuri 23756 12月 20 16:07 logfile.txt

Welcome to MinION meta-barcoding mobile laboratory!

Raw reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5 Basecalled reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/basecalling Preprocessing directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/preprocessing Reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/analysis Flow-cell: FLO-MIN106 Kit: SQK-PBK004 PCR primers trimming length: 25 bp Barcodes used in this experiment: BC01, BC02, BC03, BC04, BC05, BC06, BC07, BC08, BC09, BC10, BC11, BC12 Basecalling is going to be performed by : Guppy Basecalling Software, (C) Oxford Nanopore Technologies plc. Version 6.4.2+97a7f06, minimap2 version 2.24-r1122 Basecalling model: default Demultiplexing is going to be performed by guppy_barcoder after basecalling

The following two folders are empty.

/home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/basecalling　　 /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/preprocessing and

MaestSi commented 1 year ago

Thanks for your answer. This means base-calling did not work. Accordingly, there was nothing to demultiplex, and the following script failed due to the absence of samples to analyse. Can you please show me the command you ran? Thanks

ysuzuki1978 commented 1 year ago

I also think the problem is that it is not being passed to GUPPY well.

The command I typed in is.

. /Launch_MinION_mobile_lab.sh ~/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5/

[Edit: no need to paste the content of the script]

MaestSi commented 1 year ago

I don't think there was an error with the loading of the config file, otherwise none of the output directories would have been created. It could be that /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/basecalling directory was previously created during a test you performed, and now base-calling is being skipped since it already finds the folder. Please try to delete /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/ directory and restart the analysis.

ysuzuki1978 commented 1 year ago

I delete the specified directory and try again, guppy seems to work, but I am getting the error again. Same as before. error in evaluating the argument 'x' in selecting a method for function 'grep': invalid 'description' argument is printed at the end.

nohup.out

Welcome to MinION meta-barcoding mobile laboratory!

Raw reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5 Basecalled reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/basecalling Preprocessing directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/preprocessing Reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/analysis Flow-cell: FLO-MIN106 Kit: SQK-PBK004 PCR primers trimming length: 25 bp Barcodes used in this experiment: BC01, BC02, BC03, BC04, BC05, BC06, BC07, BC08, BC09, BC10, BC11, BC12 Basecalling is going to be performed by : Guppy Basecalling Software, (C) Oxford Nanopore Technologies plc. Version 6.4.2+97a7f06, minimap2 version 2.24-r1122 Basecalling model: default Demultiplexing is going to be performed by guppy_barcoder after basecalling

Basecalling started at Tue Dec 20 18:58:00 2022 ONT Guppy basecalling software version 6.4.2+97a7f06, minimap2 version 2.24-r1122 config file: /opt/ont/guppy/data/dna_r9.4.1_450bps_hac.cfg model file: /opt/ont/guppy/data/template_r9.4.1_450bps_hac.jsn input path: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5 save path: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/basecalling chunk size: 2000 chunks per runner: 256 minimum qscore: 9 records per file: 4000 num basecallers: 4 cpu mode: ON threads per caller: 8

Use of this software is permitted solely under the terms of the end user license agreement (EULA). By running, copying or accessing this software, you are demonstrating your acceptance of the EULA. The EULA may be found in /opt/ont/guppy/bin Found 930 input read files to process. Init time: 271 ms

0% 10 20 30 40 50 60 70 80 90 100% |----|----|----|----|----|----|----|----|----|----| Welcome to MinION meta-barcoding mobile laboratory!

Raw reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5 Basecalled reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/basecalling Preprocessing directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/preprocessing Reads directory: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/analysis Flow-cell: FLO-MIN106 Kit: SQK-PBK004 PCR primers trimming length: 25 bp Barcodes used in this experiment: BC01, BC02, BC03, BC04, BC05, BC06, BC07, BC08, BC09, BC10, BC11, BC12 Basecalling is going to be performed by : Guppy Basecalling Software, (C) Oxford Nanopore Technologies plc. Version 6.4.2+97a7f06, minimap2 version 2.24-r1122 Basecalling model: default Demultiplexing is going to be performed by guppy_barcoder after basecalling

Demultiplexing and PCR primers trimming started at Tue Dec 20 18:59:45 2022 ONT Guppy barcoding software version 6.4.2+97a7f06 input path: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/basecalling save path: /home/yakuri/nanopore/16S_rRNA_20221018/no_sample/20221018_1928_MN38820_FAV42892_85448306/fast5_analysis/preprocessing min. score front: 60 min. score rear: 60

Found 0 input files.

0% 10 20 30 40 50 60 70 80 90 100% |----|----|----|----|----|----|----|----|----|----|

Done in 9 ms. Demultiplexing and PCR primers trimming finished at Tue Dec 20 18:59:46 2022

Error in h(simpleError(msg, call)) : error in evaluating the argument 'x' in selecting a method for function 'grep': invalid 'description' argument Calls: grep -> readLines -> file -> .handleSimpleError -> h Execution halted

MaestSi commented 1 year ago

The problem is definitely guppy_basecaller not being able to base-call your reads, no idea of what could be the reason. I'd suggest checking you have read/write permissions and free space, or trying out a different Guppy version. P.s.: are you sure you do not have multiple instances of the pipeline running, thus mixing the output from different MetONTIIME_runs? The base-calling for sure started, but I see no errors or messages reporting it finished. SM

ysuzuki1978 commented 1 year ago

We checked the process with the ps command and it is denied that more than one MetONTIME is running. Changing the permissions of all MetONTIME folders to 777 does not improve the situation. There is also 800 GB of free disk space.

Are there any solutions to the following errors?

Error in h(simpleError(msg, call)) : error in evaluating the argument 'x' in selecting a method for function 'grep': invalid 'description' argument Calls: grep -> readLines -> file -> .handleSimpleError -> h Execution halted

I think it has to do with the following part of the MinION_mobile_lab.R script.

args = commandArgs(trailingOnly=TRUE)

config_file_ind <- grep(x = args, pattern = "\.R") raw_reads_ind <- setdiff(c(1, 2), config_file_ind)

MaestSi commented 1 year ago

The error that you see is just a consequence of base-calling not working. You get that error when the analysis dir does not contain any fastq files. I'd suggest checking that Guppy is properly installed and working (or testing a different version). SM

ysuzuki1978 commented 1 year ago

After re-setting the options to pass to guppy, it started working. I appreciate your detailed comments. However, PycoQC is not working properly. I will make another issue.