Rsubread crashes Rstudio while alignment
1
0
Entering edit mode
@adde7aac
Last seen 12 days ago
Switzerland

Hi everyone.

My aim was to find alternative splicing alteration between conditions of mice samples. I made a workflow with Rsubread to align and featureCounts to count. For splicing I switched to the subjunc() function. It worked well.

Then I discovered the "reportAllJunctions" argument, which I set to TRUE in the subjunc() function and received an Error "ERROR: Cigar section longer than read length: 1170268831 >= 1210, '22M164379026079M'". I though that is strange but continued without having all Junctions reported.

Some weeks later, I tried my luck again, only feeding in one sample fastq file and this time I only receive a message by Rstudio that my session was aborted due to a fatal error.

If "reportAllJunction" is set to FALSE, all works fine, also feeding into 42 sample fastq files. I am working on a Ubuntu system and should have above 60GB of RAM. Genome and GTF are both from the Gencode Website.

Below, I send you the code and the output because the alignment reached 20% but then stopped there until it crashed after a few minutes being idle.

> subjunc(index = "/home/chuddy/bioinformatics/ref_genomes/mouse_39/genome/rsubread/GRCm39",  
+       nthreads = 20, 
+       readfile1 = fastq_files_abs[1], 
+       sortReadsByCoordinates = TRUE, 
+       reportAllJunctions = T, # if T then: ERROR: Cigar section longer than read length: 1170268831 >= 1210, '22M164379026079M'
+       isGTF = T, 
+       useAnnotation = T, 
+       annot.ext = "/home/chuddy/bioinformatics/ref_genomes/mouse_39/annotation/gencode_vm34/gencode.vM34.annotation.gtf")

        ==========     _____ _    _ ____  _____  ______          _____  
        =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \ 
          =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
            ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
              ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
        ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
       Rsubread 2.14.2

//================================= setting ==================================\\
||                                                                            ||
|| Function      : Read alignment + Junction/Fusion detection (RNA-Seq)       ||
|| Input file    : 07-06h-zh_rep1_R1.fastq.gz                                 ||
|| Output file   : 07-06h-zh_rep1_R1.fastq.gz.subjunc.BAM (BAM), Sorted       ||
|| Index name    : GRCm39                                                     ||
||                                                                            ||
||                    ------------------------------------                    ||
||                                                                            ||
||                               Threads : 20                                 ||
||                          Phred offset : 33                                 ||
||                             Min votes : 1 / 28                             ||
||                        Max mismatches : 3                                  ||
||                      Max indel length : 5                                  ||
||            Report multi-mapping reads : yes                                ||
|| Max alignments per multi-mapping read : 1                                  ||
||                           Annotations : gencode.vM34.annotation.gtf (GTF)  ||
||                                                                            ||
\\============================================================================//

//=============== Running (25-Mär-2024 13:57:46, pid=128251) ================\\
||                                                                            ||
|| Check the input reads.                                                     ||
|| The input file contains base space reads.                                  ||
|| Initialise the memory objects.                                             ||
|| Estimate the mean read length.                                             ||
|| The range of Phred scores observed in the data is [2,37]                   ||
|| Create the output BAM file.                                                ||
|| Check the index.                                                           ||
|| Init the voting space.                                                     ||
|| Load the annotation file.                                                  ||
|| 863151 annotation records were loaded.                                     ||
||                                                                            ||
|| Chromosomes/contigs in index but not in annotation :                       ||
||    GL456394.1                                                              ||
||    GL456210.1                                                              ||
||    JH584295.1                                                              ||
||    GL456381.1                                                              ||
||    GL456379.1                                                              ||
||    GL456233.2                                                              ||
||    JH584299.1                                                              ||
||    GL456385.1                                                              ||
||    MU069434.1                                                              ||
||    JH584303.1                                                              ||
||    GL456366.1                                                              ||
||    GL456372.1                                                              ||
||    GL456389.1                                                              ||
||    GL456211.1                                                              ||
||    JH584296.1                                                              ||
||    GL456382.1                                                              ||
||    JH584300.1                                                              ||
||    MU069435.1                                                              ||
||    JH584304.1                                                              ||
||    GL456367.1                                                              ||
||    GL456221.1                                                              ||
||    GL456392.1                                                              ||
||    GL456354.1                                                              ||
||    GL456219.1                                                              ||
||    GL456360.1                                                              ||
||    GL456396.1                                                              ||
||    GL456212.1                                                              ||
||    JH584297.1                                                              ||
||    GL456383.1                                                              ||
||    JH584301.1                                                              ||
||    GL456370.1                                                              ||
||    GL456387.1                                                              ||
||    GL456368.1                                                              ||
||    GL456239.1                                                              ||
||    GL456378.1                                                              ||
||    GL456359.1                                                              ||
||    JH584298.1                                                              ||
||    JH584302.1                                                              ||
||    GL456390.1                                                              ||
||                                                                            ||
|| Global environment is initialised.                                         ||
|| Load the 1-th index block...                                               ||
|| The index block has been loaded.                                           ||
|| Start read mapping in chunk.                                               ||
||    0% completed, 0.6 mins elapsed, rate=214.6k reads per second            ||
||    7% completed, 0.7 mins elapsed, rate=224.4k reads per second            ||
||   13% completed, 0.8 mins elapsed, rate=220.4k reads per second            ||
||   20% completed, 0.9 mins elapsed, rate=219.0k reads per second            || 
...
HERE IT CRASHED
...
sessionInfo( ) # right before execution of subjunc()
R version 4.3.3 (2024-02-29)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 20.04.6 LTS

Matrix products: default
BLAS:   /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.9.0 
LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.9.0

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C               LC_TIME=de_CH.UTF-8        LC_COLLATE=en_US.UTF-8    
 [5] LC_MONETARY=de_CH.UTF-8    LC_MESSAGES=en_US.UTF-8    LC_PAPER=de_CH.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C             LC_MEASUREMENT=de_CH.UTF-8 LC_IDENTIFICATION=C       

time zone: Europe/Zurich
tzcode source: system (glibc)

attached base packages:
[1] grid      stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] VennDiagram_1.7.3    futile.logger_1.4.3  fgsea_1.26.0         ggrepel_0.9.5        ggforce_0.4.1       
 [6] viridis_0.6.5        viridisLite_0.4.2    ggConvexHull_0.1.0   edgeR_3.42.4         limma_3.56.2        
[11] gghighcontrast_0.1.0 Rsubread_2.14.2      lubridate_1.9.3      forcats_1.0.0        stringr_1.5.1       
[16] dplyr_1.1.4          purrr_1.0.2          readr_2.1.5          tidyr_1.3.1          tibble_3.2.1        
[21] ggplot2_3.4.4        tidyverse_2.0.0     

loaded via a namespace (and not attached):
 [1] utf8_1.2.4           generics_0.1.3       futile.options_1.0.1 stringi_1.8.3        lattice_0.22-5      
 [6] hms_1.1.3            magrittr_2.0.3       timechange_0.3.0     Matrix_1.6-5         formatR_1.14        
[11] gridExtra_2.3        fansi_1.0.6          scales_1.3.0         tweenr_2.0.2         codetools_0.2-19    
[16] cli_3.6.2            rlang_1.1.3          polyclip_1.10-6      cowplot_1.1.3        munsell_0.5.0       
[21] withr_3.0.0          parallel_4.3.3       tools_4.3.3          BiocParallel_1.34.2  tzdb_0.4.0          
[26] colorspace_2.1-0     fastmatch_1.1-4      locfit_1.5-9.8       lambda.r_1.2.4       vctrs_0.6.5         
[31] R6_2.5.1             lifecycle_1.0.4      MASS_7.3-60          pkgconfig_2.0.3      pillar_1.9.0        
[36] gtable_0.3.4         data.table_1.15.0    glue_1.7.0           Rcpp_1.0.12          xfun_0.41           
[41] tidyselect_1.2.0     rstudioapi_0.15.0    knitr_1.45           farver_2.1.1         compiler_4.3.3
Rsubjunc Rsubread • 226 views
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1
Entering edit mode
Wei Shi ★ 3.6k
@wei-shi-2183
Last seen 11 days ago
Australia/Melbourne/Olivia Newton-John …

The error associated with the reportAllJunctions parameter has been fixed in the devel version of Rsubread package. The second issue you reported is likely to be related to the first one. Please try the devel version. If the problems persist, please let us know.

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Entering edit mode

Thank for replying. I understand. Will this update be available soon in the next version and with R4.3 since the devel version needs R4.4? Because I wouldn't want to mess around with the current workflows and packages by updating my R version.

Thank you again!

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1
Entering edit mode

This update will be included in the next Bioc release scheduled next month.

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