Chuang1118
commented
2 years ago Hello,
Update zUMIs version 2.9.7c using same YAML file. It's same error! Just warning Setting locale failed is solved.
You provided these parameters:
YAML file: zUMIs_plate7.yaml
zUMIs directory: /mnt/DOSI/PMLAB/BIOINFO/scRNAseq/FB5Pseq_220414_h_huFlashseq/zUMIs/newTSO/plate7/zUMIs
STAR executable STAR
samtools executable samtools
pigz executable pigz
Rscript executable Rscript
RAM limit: null
zUMIs version 2.9.7c
mer. 18 mai 2022 18:03:46 CEST
WARNING: The STAR version used for mapping is 2.7.3a and the STAR index was created using the version 2.7.1a. This may lead to an error while mapping. If you encounter any errors at the mapping stage, please make sure to create the STAR index using STAR 2.7.3a.
Filtering...
mer. 18 mai 2022 18:04:05 CEST
[1] "441364 reads were assigned to barcodes that do not correspond to intact cells."
[1] "Found 84 daughter barcodes that can be binned into 20 parent barcodes."
[1] "Binned barcodes correspond to 31532 reads."
Mapping...
[1] "2022-05-18 18:04:10 CEST"
May 18 18:04:19 ..... started STAR run
May 18 18:04:19 ..... loading genome
May 18 18:04:19 ..... started STAR run
May 18 18:04:19 ..... loading genome
May 18 18:04:19 ..... started STAR run
May 18 18:04:19 ..... loading genome
May 18 18:06:21 ..... processing annotations GTF
May 18 18:06:21 ..... processing annotations GTF
May 18 18:06:21 ..... processing annotations GTF
May 18 18:06:37 ..... inserting junctions into the genome indices
May 18 18:06:37 ..... inserting junctions into the genome indices
May 18 18:06:37 ..... inserting junctions into the genome indices
May 18 18:10:01 ..... started 1st pass mapping
May 18 18:10:01 ..... finished 1st pass mapping
May 18 18:10:02 ..... inserting junctions into the genome indices
May 18 18:10:07 ..... started 1st pass mapping
May 18 18:10:07 ..... finished 1st pass mapping
May 18 18:10:08 ..... inserting junctions into the genome indices
May 18 18:10:17 ..... started 1st pass mapping
May 18 18:10:17 ..... finished 1st pass mapping
May 18 18:10:18 ..... inserting junctions into the genome indices
May 18 18:11:40 ..... started mapping
May 18 18:11:41 ..... finished mapping
May 18 18:11:42 ..... finished successfully
May 18 18:11:44 ..... started mapping
May 18 18:11:45 ..... finished mapping
May 18 18:11:46 ..... finished successfully
May 18 18:11:57 ..... started mapping
May 18 18:11:57 ..... finished mapping
May 18 18:11:59 ..... finished successfully
mer. 18 mai 2022 18:12:00 CEST
Counting...
[1] "2022-05-18 18:12:10 CEST"
[1] "4.5e+08 Reads per chunk"
[1] "Loading reference annotation from:"
[1] "/mnt/DOSI/PMLAB/BIOINFO/scRNAseq/FB5Pseq_220414_h_huFlashseq/zUMIs/newTSO/plate7/FLASHfb5p.final_annot.gtf"
[1] "Annotation loaded!"
Warning message:
`as_quosure()` requires an explicit environment as of rlang 0.3.0.
Please supply `env`.
This warning is displayed once per session.
[1] "Assigning reads to features (ex)"
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 1.32.4
//========================== featureCounts setting ===========================\\
|| ||
|| Input files : 1 BAM file ||
|| S FLASHfb5p.filtered.tagged.Aligned.out.bam ||
|| ||
|| Annotation : R data.frame ||
|| Assignment details : <input_file>.featureCounts.bam ||
|| (Note that files are saved to the output directory) ||
|| ||
|| Dir for temp files : . ||
|| Threads : 30 ||
|| Level : meta-feature level ||
|| Paired-end : yes ||
|| Multimapping reads : counted ||
|| Multiple alignments : primary alignment only ||
|| Multi-overlapping reads : not counted ||
|| Min overlapping bases : 1 ||
|| ||
|| Chimeric reads : not counted ||
|| Both ends mapped : not required ||
|| ||
\\===================== http://subread.sourceforge.net/ ======================//
//================================= Running ==================================\\
|| ||
|| Load annotation file .Rsubread_UserProvidedAnnotation_pid491710 ... ||
|| Features : 328745 ||
|| Meta-features : 60761 ||
|| Chromosomes/contigs : 151 ||
|| ||
|| Process BAM file FLASHfb5p.filtered.tagged.Aligned.out.bam... ||
|| Single-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 0 ||
|| Successfully assigned alignments : 0 ||
|| Running time : 0.00 minutes ||
|| ||
|| ||
\\===================== http://subread.sourceforge.net/ ======================//
[1] "Assigning reads to features (in)"
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 1.32.4
//========================== featureCounts setting ===========================\\
|| ||
|| Input files : 1 BAM file ||
|| S FLASHfb5p.filtered.tagged.Aligned.out.bam. ... ||
|| ||
|| Annotation : R data.frame ||
|| Assignment details : <input_file>.featureCounts.bam ||
|| (Note that files are saved to the output directory) ||
|| ||
|| Dir for temp files : . ||
|| Threads : 30 ||
|| Level : meta-feature level ||
|| Paired-end : yes ||
|| Multimapping reads : counted ||
|| Multiple alignments : primary alignment only ||
|| Multi-overlapping reads : not counted ||
|| Min overlapping bases : 1 ||
|| ||
|| Chimeric reads : not counted ||
|| Both ends mapped : not required ||
|| ||
\\===================== http://subread.sourceforge.net/ ======================//
//================================= Running ==================================\\
|| ||
|| Load annotation file .Rsubread_UserProvidedAnnotation_pid491710 ... ||
|| Features : 232472 ||
|| Meta-features : 27187 ||
|| Chromosomes/contigs : 34 ||
|| ||
|| Process BAM file FLASHfb5p.filtered.tagged.Aligned.out.bam.ex.featureC ... ||
|| Single-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 0 ||
|| Successfully assigned alignments : 0 ||
|| Running time : 0.00 minutes ||
|| ||
|| ||
\\===================== http://subread.sourceforge.net/ ======================//
[1] "2022-05-18 18:13:33 CEST"
[1] "Coordinate sorting final bam file..."
[1] "2022-05-18 18:13:34 CEST"
[1] "Here are the detected subsampling options:"
[1] "Automatic downsampling"
[1] "Working on barcode chunk 1 out of 1"
[1] "Processing 23 barcodes in this chunk..."
Error in .checkTypos(e, names_x) : Object 'GEin' not found amongst ftype
Calls: reads2genes_new ... tryCatchList -> tryCatchOne -> <Anonymous> -> .checkTypos
Execution halted
mer. 18 mai 2022 18:13:38 CEST
Loading required package: yaml
Loading required package: Matrix
[1] "loomR found"
Error in gzfile(file, "rb") : cannot open the connection
Calls: rds_to_loom -> readRDS -> gzfile
In addition: Warning message:
In gzfile(file, "rb") :
cannot open compressed file '/mnt/DOSI/PMLAB/BIOINFO/scRNAseq/FB5Pseq_220414_h_huFlashseq/zUMIs/newTSO/plate7/zUMIs_output/expression/FLASHfb5p.dgecounts.rds', probable reason 'No such file or directory'
Execution halted
mer. 18 mai 2022 18:13:41 CEST
Descriptive statistics...
[1] "I am loading useful packages for plotting..."
[1] "2022-05-18 18:13:42 CEST"
Error in gzfile(file, "rb") : cannot open the connection
Calls: readRDS -> gzfile
In addition: Warning message:
In gzfile(file, "rb") :
cannot open compressed file '/mnt/DOSI/PMLAB/BIOINFO/scRNAseq/FB5Pseq_220414_h_huFlashseq/zUMIs/newTSO/plate7/zUMIs_output/expression/FLASHfb5p.dgecounts.rds', probable reason 'No such file or directory'
Execution halted
mer. 18 mai 2022 18:13:52 CEST
cziegenhain
Hello, zUMIs team,
I a new zUMIs user, I have faced the problem by using zUMIs in mapping step. Install zUMIs For reproducible reason, I have used singularity V3 from docker hub docker://tomkellygenetics/zumis:v2.9.7 Input zcat FLASH_1GC_P7_S7L001R1_001.fastq.gz | head # my read1 is cDNA
zcat FLASH_1GC_P7_S7L001R2_001.fastq.gz | head # my read2 is BC+UMI
CMD
zUMIs part
Both FLASHfb5p.filtered.Aligned.GeneTagged.sorted.bam and FLASHfb5p.filtered.tagged.Aligned.out.bam is empty, in other word, only have header. samtools view FLASHfb5p.filtered.tagged.unmapped.bam | head -n 5
samtools view FLASHfb5p.filtered.tagged.bam | head -n 5
The YAML file and full standard output zUMIs_plate7.yaml nohup.out Here, I got a lot of warning Setting locale failed. I can't handle it.
Screenshots issue
Additional message Would you provide a singularity image more recently?
Thanks, Chuang