Nohup failed to run command '/.': Permission denied

Question

[Christopher]

Hello guys!

I'm going to introduce myself, so please excuse any errors in my English. My name is Christopher Lee, and I'm Brazilian.

I have been facing a problem for about two weeks that I've been trying to fix. Initially, I was attempting to use an Oracle VM to run the bcl2fastq2 software, as shown in the first image. I contacted Illumina, and they informed me that I needed to use BCL convert to convert my cbcl files because (I believe) they want to transition from bcl2fastq2 to BCL convert. So, I followed their advice and made the switch.

Now, I'm encountering another issue. Since I am using a VM, I installed CentOS7 on it to utilize it. However, when I try to run the code, I receive the following error message:

"nohup: ignoring input and appending output to ‘nohup.out’ nohup: failed to run command ‘/usr/local/bin/bcl-convert’: Permission denied"

I have attempted various solutions to resolve this problem, but I am now unsure of how to fix it. If any of you can assist me, I would greatly appreciate it.

Ahhh, I'm working with Novaseq 6000, and it gives me the .cbcl file instead of the .bcl file.

[Pierre Lindenbaum]

please, copy-n-paste the text instead of using screenshots (text is searchable, carbon footprint etc...). show us the original command. furthermore, you should avoid nohup, use screen/tmux instead (see The .bcf file generated by bcftools mpileup cannot be opened with BCFtools )

Answer 1

[GenoMax]

In spite of talking about bcl-convert you appear to be still using bcl2fastq for this conversion in the screenshot. This is fine to do for NovaSeq 6000.

It appears that you are missing at least one (C)BCL (cbcl files contain aggregated BCL data) file in the first cycle folder. This can happen at times. You can get past this error using --ignore-missing-bcls option for bcl2fastq with your command. This option will replace the missing base with a N call. Hopefully there are no additional bcl files missing. If you encounter a lot of missing files you will want to check the original data folder (in case you made a copy that you are working with). Too many missing BCL files will result in a lot of missing data in cycles.

---

As for your problem with /usr/local/bin/bcl-convert, have you made sure that the program is executable? Simply doing chmod +x /usr/local/bin/bcl-convert should fix the permissions, if that is the issue.

[Christopher]

GenoMax, I kind of found the problem. I was using an older version of bcl2fastq2 (version 2.18) instead of version 2.20.

However, now I've encountered another issue. After running the bcl2fastq command, I can't find the .cbcl files as I'll demonstrate below.

2023-07-23 16:30:27 [229f880] Command-line invocation: bcl2fastq --ignore-missing-bcls
2023-07-23 16:30:27 [229f880] INFO: Minimum log level: INFO
2023-07-23 16:30:27 [229f880] INFO: Sample sheet: '/home/vboxuser/Desktop/bcl2fastq2-v2-20-0-linux-x86-64/usr/local/bin/teste-20230723T135530Z-001/teste/SampleSheet.csv'
2023-07-23 16:30:27 [229f880] INFO: Sample sheet: NOT FOUND
2023-07-23 16:30:27 [229f880] INFO: Runfolder path: '/home/vboxuser/Desktop/bcl2fastq2-v2-20-0-linux-x86-64/usr/local/bin/teste-20230723T135530Z-001/teste'
2023-07-23 16:30:27 [229f880] INFO: Input path: '/home/vboxuser/Desktop/bcl2fastq2-v2-20-0-linux-x86-64/usr/local/bin/teste-20230723T135530Z-001/teste/Data/Intensities/BaseCalls/'
2023-07-23 16:30:27 [229f880] INFO: Intensities path: '/home/vboxuser/Desktop/bcl2fastq2-v2-20-0-linux-x86-64/usr/local/bin/teste-20230723T135530Z-001/teste/Data/Intensities/'
2023-07-23 16:30:27 [229f880] INFO: Output path: '/home/vboxuser/Desktop/bcl2fastq2-v2-20-0-linux-x86-64/usr/local/bin/teste-20230723T135530Z-001/teste/Data/Intensities/BaseCalls/'
2023-07-23 16:30:27 [229f880] INFO: InterOp path: '/home/vboxuser/Desktop/bcl2fastq2-v2-20-0-linux-x86-64/usr/local/bin/teste-20230723T135530Z-001/teste/InterOp/'
2023-07-23 16:30:27 [229f880] INFO: Stats path: '/home/vboxuser/Desktop/bcl2fastq2-v2-20-0-linux-x86-64/usr/local/bin/teste-20230723T135530Z-001/teste/Data/Intensities/BaseCalls/Stats/'
2023-07-23 16:30:27 [229f880] INFO: Reports path: '/home/vboxuser/Desktop/bcl2fastq2-v2-20-0-linux-x86-64/usr/local/bin/teste-20230723T135530Z-001/teste/Data/Intensities/BaseCalls/Reports/'
2023-07-23 16:30:27 [229f880] INFO: Detected CPUs: 3
2023-07-23 16:30:27 [229f880] INFO: Loading threads: 4
2023-07-23 16:30:27 [229f880] INFO: Processing threads: 3
2023-07-23 16:30:27 [229f880] INFO: Writing threads: 4
2023-07-23 16:30:27 [229f880] INFO: Allowed barcode mismatches: 1
2023-07-23 16:30:27 [229f880] INFO: Tiles: <ALL>
2023-07-23 16:30:27 [229f880] INFO: Minimum trimmed read length: 35
2023-07-23 16:30:27 [229f880] INFO: Use bases masks: <NONE>
2023-07-23 16:30:27 [229f880] INFO: Mask short adapter reads: 22
2023-07-23 16:30:27 [229f880] INFO: Adapter stringency: 0.9
2023-07-23 16:30:27 [229f880] INFO: Adapter trimming method: Allow matches with indels
2023-07-23 16:30:27 [229f880] INFO: Ignore missing BCLs: YES
2023-07-23 16:30:27 [229f880] INFO: Ignore missing filters: NO
2023-07-23 16:30:27 [229f880] INFO: Ignore missing positions: NO
2023-07-23 16:30:27 [229f880] INFO: Ignore missing controls: NO
2023-07-23 16:30:27 [229f880] INFO: Include non-PF clusters: NO
2023-07-23 16:30:27 [229f880] INFO: Create FASTQs for index reads: NO
2023-07-23 16:30:27 [229f880] INFO: Use bgzf compression for FASTQ files: YES
2023-07-23 16:30:27 [229f880] INFO: FASTQ compression level: 4
2023-07-23 16:30:27 [229f880] INFO: RunInfo.xml: '"/home/vboxuser/Desktop/bcl2fastq2-v2-20-0-linux-x86-64/usr/local/bin/teste-20230723T135530Z-001/teste/RunInfo.xml"'
2023-07-23 16:30:27 [229f880] INFO: Lane: 1
2023-07-23 16:30:27 [229f880] INFO: Mask:
2023-07-23 16:30:27 [229f880] INFO:   Sample: #0 'unknown' 'Undetermined' [default]
2023-07-23 16:30:27 [229f880] INFO: Read: 1 data read
2023-07-23 16:30:27 [229f880] INFO:   Cycles: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151
2023-07-23 16:30:27 [229f880] INFO: Read: 1 index read
2023-07-23 16:30:27 [229f880] INFO:   Cycles: 152,153,154,155,156,157,158,159,160,161
2023-07-23 16:30:27 [229f880] INFO: Read: 2 index read
2023-07-23 16:30:27 [229f880] INFO:   Cycles: 162,163,164,165,166,167,168,169,170,171
2023-07-23 16:30:27 [229f880] INFO: Read: 2 data read
2023-07-23 16:30:27 [229f880] INFO:   Cycles: 172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322
2023-07-23 16:30:27 [229f880] INFO: Lane: 2
2023-07-23 16:30:27 [229f880] INFO: Mask:
2023-07-23 16:30:27 [229f880] INFO:   Sample: #0 'unknown' 'Undetermined' [default]
2023-07-23 16:30:27 [229f880] INFO: Read: 1 data read
2023-07-23 16:30:27 [229f880] INFO:   Cycles: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151
2023-07-23 16:30:27 [229f880] INFO: Read: 1 index read
2023-07-23 16:30:27 [229f880] INFO:   Cycles: 152,153,154,155,156,157,158,159,160,161
2023-07-23 16:30:27 [229f880] INFO: Read: 2 index read
2023-07-23 16:30:27 [229f880] INFO:   Cycles: 162,163,164,165,166,167,168,169,170,171
2023-07-23 16:30:27 [229f880] INFO: Read: 2 data read
2023-07-23 16:30:27 [229f880] INFO:   Cycles: 172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322
2023-07-23 16:30:27 [229f880] INFO: Patterned flowcell detected
2023-07-23 16:30:27 [229f880] INFO:   Tile: 1101 (index: 0, skipped tiles: 0)
2023-07-23 16:30:27 [229f880] INFO:   Tile: 1102 (index: 1, skipped tiles: 0)
2023-07-23 16:30:27 [229f880] INFO:   Tile: 1103 (index: 2, skipped tiles: 0)
2023-07-23 16:30:27 [229f880] INFO:   Tile: 1104 (index: 3, skipped tiles: 0)
2023-07-23 16:30:27 [229f880] INFO:   Tile: 1105 (index: 4, skipped tiles: 0)
2023-07-23 16:30:27 [229f880] INFO:   Tile: 1106 (index: 5, skipped tiles: 0)
......
2023-07-23 16:30:27 [229f880] INFO:   Tile: 2276 (index: 309, skipped tiles: 0)
2023-07-23 16:30:27 [229f880] INFO:   Tile: 2277 (index: 310, skipped tiles: 0)
2023-07-23 16:30:27 [229f880] INFO:   Tile: 2278 (index: 311, skipped tiles: 0)
2023-07-23 16:30:27 [229f880] WARNING: No cbcl files found for cycle 1. Attempt 1 of 3. Retry with cycle 2.
2023-07-23 16:30:27 [229f880] WARNING: No cbcl files found for cycle 2. Attempt 2 of 3. Retry with cycle 3.
2023-07-23 16:30:27 [229f880] ERROR: bcl2fastq::common::Exception: 2023-Jul-23 16:30:27: Success (0): /TeamCityBuildAgent/work/556afd631a5b66d8/src/cxx/lib/layout/Layout.cpp(1028): Throw in function static void bcl2fastq::layout::TileLayoutDetector::readTilesFromCbclHeaders(const boost::filesystem::path&, const bcl2fastq::config::SampleSheetCsv&, const std::vector<boost::basic_regex<char, boost::regex_traits<char> > >&, const string&, std::vector<bcl2fastq::layout::LaneInfo>&, bcl2fastq::common::TileFileMap&, bcl2fastq::common::NumBasesPerByte&, bcl2fastq::common::CycleNumber, bcl2fastq::common::CycleNumber, bool)
Dynamic exception type: boost::exception_detail::clone_impl<bcl2fastq::common::InputDataError>
std::exception::what:
No cbcl files found for cycle 3. Attempt 3 of 3.
Corrupt or missing .cbcl files were found for 3 consecutive cycles, verify the source of the data.

[GenoMax]

Nothing should be happening to the cbcl files since all data output is written to a different output directory (specified by -o option).

Here is the issue.

Corrupt or missing .cbcl files were found for 3 consecutive cycles, verify the source of the data.

Looks like you are also missing bcl files for other cycles or they are corrupt. I was afraid of this. It is possible that either your copy of this data is corrupt or this run has suffered additional issues.

Are you running the sequencer yourself or did you get this data from some other provider? They should really be doing this for you if latter is true.

[Christopher]

Yeah! That's what happened. I only got a few CBCL files to test the bcl2fastq2. I'm kind of a newbie with these things, and I didn't know that I needed the full .cbcl files.

So, to fix the problem with the .cbcl files, I needed to update from version 2.18 to version 2.20, and all .cbcl files.

GenoMax, thank you for your support!