Error in (function (cl, name, valueClass) :

[ytang0831]

Hi， I recently had a problem I couldn't solve，Judging by the results, there seems to be something wrong with matrix, but Featurecounts worked fine. At the same time I noticed that there was no output file named featurecounts.bam，really confusing. Looking forward to your answer!

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Good news! A newer version of zUMIs is available at https://github.com/sdparekh/zUMIs

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You provided these parameters: YAML file: zUMIs-single_1_4.yaml zUMIs directory: /share/home/fanglab/tangyin/HSET/1.rawdata/RNA/zUMIs-master STAR executable STAR samtools executable samtools pigz executable pigz Rscript executable Rscript RAM limit: null zUMIs version 2.7.1c

2021年 12月 03日 星期五 14:36:33 CST Counting... [1] "2021-12-03 14:37:35 CST" [1] "2e+09 Reads per chunk" [1] "Loading reference annotation from:" [1] "/share/home/fanglab/tangyin/HSET/1.rawdata/RNA/result/Exp_HSET_Single_cell_rep1_4/HSET-single1_4.final_annot.gtf" [1] "Annotation loaded!" [1] "Assigning reads to features (ex)"

    ==========     _____ _    _ ____  _____  ______          _____  
    =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \ 
      =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
        ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
          ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
    ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
   Rsubread 1.32.4

//========================== featureCounts setting ===========================\						Input files : 1 BAM file				S HSET-single1_4.filtered.tagged.Aligned.out ...
Annotation : R data.frame
Assignment details : .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_pid81819 ...				Features : 233760				Meta-features : 22813				Chromosomes/contigs : 32
Process BAM file HSET-single1_4.filtered.tagged.Aligned.out.bam...
Single-end reads are included.
Assign alignments to features...
Total alignments : 4364758
Successfully assigned alignments : 625181 (14.3%)
Running time : 0.11 minutes

\===================== http://subread.sourceforge.net/ ======================//

[1] "Coordinate sorting final bam file..." [bam_sort_core] merging from 0 files and 30 in-memory blocks... [1] "Here are the detected subsampling options:" [1] "Automatic downsampling" [1] "Extracting reads from bam file(s)..." [1] "Working on barcode chunk 1 out of 1" [1] "Processing 4 barcodes in this chunk..." Error in (function (cl, name, valueClass) : assignment of an object of class “numeric” is not valid for @‘Dim’ in an object of class “dgTMatrix”; is(value, "integer") is not TRUE Calls: convert2countM -> .makewide -> ->

data.table and Rsubread version： Rsubread_2.4.3 data.table_1.14.0

run_log here: You provided these parameters: YAML file: zUMIs-single_1_4.yaml zUMIs directory: /share/home/fanglab/tangyin/HSET/1.rawdata/RNA/zUMIs-master STAR executable STAR samtools executable samtools pigz executable pigz Rscript executable Rscript RAM limit: null zUMIs version 2.7.1c

[ytang0831]

1400 -rw-r--r-- 1 tangyin fanglab 1432669 12月 3 10:59 HSET-single1_4.BCstats.txt 308593 -rw-r--r-- 1 tangyin fanglab 315998480 12月 3 14:39 HSET-single1_4.filtered.Aligned.GeneTagged.sorted.bam 2064 -rw-r--r-- 1 tangyin fanglab 2112720 12月 3 14:39 HSET-single1_4.filtered.Aligned.GeneTagged.sorted.bam.bai 378849 -rw-r--r-- 1 tangyin fanglab 387941033 12月 3 11:08 HSET-single1_4.filtered.tagged.Aligned.out.bam 2 -rw-r--r-- 1 tangyin fanglab 1851 12月 3 11:08 HSET-single1_4.filtered.tagged.Log.final.out 570 -rw-r--r-- 1 tangyin fanglab 583466 12月 3 11:08 HSET-single1_4.filtered.tagged.Log.out 1 -rw-r--r-- 1 tangyin fanglab 447 12月 3 11:08 HSET-single1_4.filtered.tagged.Log.progress.out 1 -rw-r--r-- 1 tangyin fanglab 434 12月 3 11:08 HSET-single1_4.filtered.tagged.Log.std.out 222 -rw-r--r-- 1 tangyin fanglab 226549 12月 3 11:08 HSET-single1_4.filtered.tagged.SJ.out.tab 8 drwx------ 2 tangyin fanglab 8192 12月 3 11:01 HSET-single1_4.filtered.tagged._STARgenome 8 drwx------ 2 tangyin fanglab 8192 12月 3 11:04 HSET-single1_4.filtered.tagged._STARpass1 8 drwx------ 2 tangyin fanglab 8192 12月 3 11:08 HSET-single1_4.filtered.tagged._STARtmp 323668 -rw-r--r-- 1 tangyin fanglab 331435781 12月 3 11:00 HSET-single1_4.filtered.tagged.unmapped.bam 147853 -rwxr-xr-x 1 tangyin fanglab 151400465 12月 3 11:00 HSET-single1_4.final_annot.gtf 1 -rw-r--r-- 1 tangyin fanglab 297 12月 3 14:36 HSET-single1_4.zUMIs_runlog.txt 8 drwxr-xr-x 5 tangyin fanglab 8192 12月 3 14:39 zUMIs_output

Bam files are not empty and can be opened with smatools View

[cziegenhain]

Hi,

I don't see your YAML file so cant check if there is any obvious errors coming from that. It seems from your output that there are only 4 barcodes to be processed? Is that expected? The gene assignment rate seems very low too, I could imagine that there are matrix creation errors if some of these barcodes have 0 counts assigned or something like that.

Best, Christoph

[ytang0831]

Thanks, @cziegenhain . I have fixed this problem.This problem is most likely caused by incompatibility between R (4.0) and zUMIs version 2.7. When I switched to the R3.6 environment and added the -c parameter, it worked