Can the correctSystematicG = T parameter be used for a BAM file generated from STAR mapping of paired-end reads

[QiliShi]

Hello,

I encountered an issue while using the correctSystematicG = T parameter with a BAM file generated from paired-end reads mapped with STAR.

R code:

bamfile <-CAGEexp(genomeName="BSgenome.Hsapiens.UCSC.hg38",inputFiles='SRR7092026Aligned.sortedByCoord.out.bam',inputFilesType="bamPairedEnd",sampleLabels='example')
ce <- getCTSS(bamfile)
Error message:


Reading in file: ~/Intergetic_transcripts/cage_seq/alignment/SRR7092026Aligned.sortedByCoord.out.bam...
        -> Filtering out low quality reads...
Loading required namespace: BSgenome.Hsapiens.UCSC.hg38
        -> Removing the first base of the reads if 'G' and not aligned to the genome...
        -> Estimating the frequency of adding a 'G' nucleotide and correcting the systematic bias...
Error: BiocParallel errors
  1 remote errors, element index: 1
  0 unevaluated and other errors
  first remote error:
Error in setnames(CTSS, c("chr", "pos", "strand", sample.label)): Can't assign 4 names to a 0 column data.table

It works fine using the correctSystematicG = F
ce <- getCTSS(bamfile,correctSystematicG = F)

Reading in file: ~/Intergetic_transcripts/cage_seq/alignment/SRR7092026Aligned.sortedByCoord.out.bam...
-> Filtering out low quality reads...
-> Removing the first base of the reads if 'G' and not aligned to the genome...

R4.3.2
attached packages:
[1] CAGEr_2.8.0 MultiAssayExperiment_1.28.0
[3] SummarizedExperiment_1.32.0 Biobase_2.62.0
[5] GenomicRanges_1.54.1 GenomeInfoDb_1.38.1
[7] IRanges_2.36.0 S4Vectors_0.40.2
[9] BiocGenerics_0.48.1 MatrixGenerics_1.14.0
[11] matrixStats_1.2.0

loaded via a namespace (and not attached):
[1] RColorBrewer_1.1-3 rstudioapi_0.15.0
[3] magrittr_2.0.3 GenomicFeatures_1.54.1
[5] rmarkdown_2.25 BiocIO_1.12.0
[7] zlibbioc_1.48.0 vctrs_0.6.5
[9] memoise_2.0.1 Rsamtools_2.18.0
[11] DelayedMatrixStats_1.24.0 RCurl_1.98-1.14
[13] base64enc_0.1-3 BiocBaseUtils_1.4.0
[15] htmltools_0.5.7 S4Arrays_1.2.0
[17] progress_1.2.3 curl_5.1.0
[19] SparseArray_1.2.2 Formula_1.2-5
[21] KernSmooth_2.23-22 htmlwidgets_1.6.4
[23] plyr_1.8.9 Gviz_1.46.1
[25] cachem_1.0.8 GenomicAlignments_1.38.0
[27] lifecycle_1.0.4 pkgconfig_2.0.3
[29] Matrix_1.6-5 R6_2.5.1
[31] fastmap_1.1.1 GenomeInfoDbData_1.2.11
[33] digest_0.6.34 colorspace_2.1-0
[35] AnnotationDbi_1.64.1 Hmisc_5.1-1
[37] RSQLite_2.3.4 vegan_2.6-4
[39] filelock_1.0.3 fansi_1.0.6
[41] mgcv_1.9-1 httr_1.4.7
[43] abind_1.4-5 compiler_4.3.2
[45] bit64_4.0.5 htmlTable_2.4.2
[47] backports_1.4.1 CAGEfightR_1.22.0
[49] BiocParallel_1.36.0 DBI_1.2.1
[51] biomaRt_2.58.0 MASS_7.3-60
[53] rappdirs_0.3.3 DelayedArray_0.28.0
[55] rjson_0.2.21 permute_0.9-7
[57] gtools_3.9.5 tools_4.3.2
[59] foreign_0.8-86 nnet_7.3-19
[61] glue_1.7.0 restfulr_0.0.15
[63] nlme_3.1-164 stringdist_0.9.10
[65] grid_4.3.2 checkmate_2.3.0
[67] reshape2_1.4.4 cluster_2.1.6
[69] generics_0.1.3 operator.tools_1.6.3
[71] gtable_0.3.4 BSgenome_1.70.1
[73] formula.tools_1.7.1 ensembldb_2.26.0
[75] data.table_1.14.10 hms_1.1.3
[77] xml2_1.3.6 utf8_1.2.4
[79] XVector_0.42.0 pillar_1.9.0
[81] stringr_1.5.1 splines_4.3.2
[83] dplyr_1.1.4 BiocFileCache_2.10.1
[85] lattice_0.22-5 rtracklayer_1.62.0
[87] bit_4.0.5 deldir_2.0-2
[89] biovizBase_1.50.0 BSgenome.Hsapiens.UCSC.hg38_1.4.5
[91] tidyselect_1.2.0 Biostrings_2.70.1
[93] knitr_1.45 gridExtra_2.3
[95] ProtGenerics_1.34.0 xfun_0.41
[97] stringi_1.8.3 VGAM_1.1-9
[99] lazyeval_0.2.2 yaml_2.3.8
[101] som_0.3-5.1 evaluate_0.23
[103] codetools_0.2-19 interp_1.1-5
[105] tibble_3.2.1 cli_3.6.2
[107] rpart_4.1.23 munsell_0.5.0
[109] dichromat_2.0-0.1 Rcpp_1.0.12
[111] dbplyr_2.4.0 png_0.1-8
[113] XML_3.99-0.16 parallel_4.3.2
[115] ggplot2_3.4.4 assertthat_0.2.1
[117] blob_1.2.4 prettyunits_1.2.0
[119] latticeExtra_0.6-30 jpeg_0.1-10
[121] AnnotationFilter_1.26.0 sparseMatrixStats_1.14.0
[123] bitops_1.0-7 VariantAnnotation_1.48.1
[125] scales_1.3.0 crayon_1.5.2
[127] rlang_1.1.3 KEGGREST_1.42.0

[charles-plessy]

Hello, can you share your BAM file with me?

[QiliShi]

Thank you for your prompt reply. I've just sent the BAM file as an attachment by email.

[QiliShi]

Hello, it is plausible that the absence of sequencing quality information in the downloaded FASTQ file may indeed be the root cause of the observed issue. Unfortunately, I am unable to verify this assertion as I do not currently have access to my own cage-seq data for comparison or validation purposes