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VCCRI / Sierra

Discover differential transcript usage from polyA-captured single cell RNA-seq data
GNU General Public License v3.0
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Error in CountPeaks step #41

Closed johnchamberlin closed 3 years ago

johnchamberlin commented 3 years ago

I'm attempting to figure out the source of the following error: Error in (function (classes, fdef, mtable) : unable to find an inherited method for function 'writeMM' for signature '"NULL"'

I just re-installed Sierra after finding the same error described here: https://github.com/VCCRI/Sierra/issues/35 I have previously ran Sierra successfully on 10x data from the same pipeline (STARsolo). The current dataset is substantially larger, about 1.5 billion reads per sample. Running via slurm with 128GB RAM provided.

Edit: subsetting the BAM to chr19 only did not resolve the error so I don't think this is a memory issue.

Command:

CountPeaks(peak.sites.file = peak.output.file, # FindPeaks runs fine gtf.file = gtf, # ensembl build 99 gtf for mouse, filterd per CellRanger spec bamfile = bam, # indexed bam file from STARsolo whitelist.file = bcs, # text file containing one column with barcode sequences output.dir = count.dir, countUMI = TRUE, chr.names = c("1","2","3"), # subset to three chromosomes to expedite testing filter.chr=TRUE, # (your documentation describes this option incorrectly) ncores = 8)

Full output below:

Warning messages: 1: replacing previous import 'GenomicRanges::union' by 'dplyr::union' when loading 'Sierra' 2: replacing previous import 'GenomicRanges::intersect' by 'dplyr::intersect' when loading 'Sierra' 3: replacing previous import 'GenomicRanges::setdiff' by 'dplyr::setdiff' when loading 'Sierra' 4: replacing previous import 'Gviz::tail' by 'utils::tail' when loading 'Sierra' 5: replacing previous import 'Gviz::head' by 'utils::head' when loading 'Sierra' There are 8266 whitelist barcodes. Import genomic features from the file as a GRanges object ... OK Prepare the 'metadata' data frame ... OK Make the TxDb object ... OK There are 168884 sites Doing counting for each site... Processing chr: 3 and strand 1 Processing chr: 2 and strand 1 Processing chr: 1 and strand 1 and strand -1 and strand -1 and strand -1 Error in (function (classes, fdef, mtable) : unable to find an inherited method for function 'writeMM' for signature '"NULL"' Calls: CountPeaks -> -> In addition: Warning message: In .get_cds_IDX(mcols0$type, mcols0$phase) : The "phase" metadata column contains non-NA values for features of type stop_codon. This information was ignored. Execution halted

thank you,

johnchamberlin commented 3 years ago

sessionInfo() R version 4.0.2 (2020-06-22) Platform: x86_64-pc-linux-gnu (64-bit) Running under: CentOS Linux 7 (Core)

Matrix products: default BLAS/LAPACK: /uufs/chpc.utah.edu/sys/installdir/intel/compilers_and_libraries_2019.5.281/linux/mkl/lib/intel64_lin/libmkl_intel_lp64.so

locale: [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8 [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8 [7] LC_PAPER=en_US.UTF-8 LC_NAME=C [9] LC_ADDRESS=C LC_TELEPHONE=C [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C

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

other attached packages: [1] Sierra_0.99.27

loaded via a namespace (and not attached): [1] ProtGenerics_1.20.0 bitops_1.0-6 [3] matrixStats_0.56.0 bit64_0.9-7 [5] RColorBrewer_1.1-2 progress_1.2.2 [7] httr_1.4.1 GenomeInfoDb_1.24.2 [9] tools_4.0.2 backports_1.1.8 [11] R6_2.4.1 rpart_4.1-15 [13] lazyeval_0.2.2 Hmisc_4.4-0 [15] DBI_1.1.0 BiocGenerics_0.34.0 [17] Gviz_1.32.0 colorspace_1.4-1 [19] nnet_7.3-14 tidyselect_1.1.0 [21] gridExtra_2.3 prettyunits_1.1.1 [23] bit_1.1-15.2 curl_4.3 [25] compiler_4.0.2 Biobase_2.48.0 [27] htmlTable_2.0.1 DelayedArray_0.14.0 [29] rtracklayer_1.48.0 scales_1.1.1 [31] checkmate_2.0.0 genefilter_1.70.0 [33] askpass_1.1 rappdirs_0.3.1 [35] stringr_1.4.0 digest_0.6.25 [37] Rsamtools_2.4.0 foreign_0.8-80 [39] XVector_0.28.0 dichromat_2.0-0 [41] base64enc_0.1-3 jpeg_0.1-8.1 [43] pkgconfig_2.0.3 htmltools_0.5.0 [45] ensembldb_2.12.1 dbplyr_1.4.4 [47] BSgenome_1.56.0 htmlwidgets_1.5.1 [49] rlang_0.4.7 rstudioapi_0.11 [51] RSQLite_2.2.0 generics_0.0.2 [53] BiocParallel_1.22.0 acepack_1.4.1 [55] dplyr_1.0.0 VariantAnnotation_1.34.0 [57] RCurl_1.98-1.2 magrittr_1.5 [59] GenomeInfoDbData_1.2.3 Formula_1.2-3 [61] Matrix_1.2-18 Rcpp_1.0.5 [63] munsell_0.5.0 S4Vectors_0.26.1 [65] lifecycle_0.2.0 stringi_1.4.6 [67] SummarizedExperiment_1.18.2 zlibbioc_1.34.0 [69] BiocFileCache_1.12.0 grid_4.0.2 [71] blob_1.2.1 parallel_4.0.2 [73] crayon_1.3.4 lattice_0.20-41 [75] Biostrings_2.56.0 splines_4.0.2 [77] annotate_1.66.0 GenomicFeatures_1.40.1 [79] hms_0.5.3 knitr_1.33 [81] pillar_1.4.6 GenomicRanges_1.40.0 [83] codetools_0.2-16 biomaRt_2.44.1 [85] stats4_4.0.2 XML_3.99-0.4 [87] glue_1.4.1 biovizBase_1.36.0 [89] latticeExtra_0.6-29 data.table_1.12.8 [91] foreach_1.5.0 png_0.1-7 [93] vctrs_0.3.1 gtable_0.3.0 [95] openssl_1.4.2 purrr_0.3.4 [97] assertthat_0.2.1 ggplot2_3.3.2 [99] xfun_0.24 xtable_1.8-4 [101] AnnotationFilter_1.12.0 survival_3.1-12 [103] SingleCellExperiment_1.10.1 tibble_3.0.3 [105] iterators_1.0.12 GenomicAlignments_1.24.0 [107] AnnotationDbi_1.50.1 memoise_1.1.0 [109] IRanges_2.22.2 cluster_2.1.0 [111] ellipsis_0.3.1

johnchamberlin commented 3 years ago

I suspect the problem is that the CB and UMI tags were not included in the STARsolo outputted bam. (--outSAMattributes ... option was omitted, defaults to no tags in BAM). I'll reopen if this doesn't fix it. thanks,