So I have been trying for the past 4 days to run icetea to detect TSS out of my .bam files. My fastq files were aligned using STAR and PCR duplicates were also removed using STAR. However, for some reason, when I use the files to detect TSS the scores come back as NA and I don't know how to fix this. Please help.

This is what I got so far:

Codes:

files <- list.files(path = ".", pattern = ".bam$", full.names = TRUE) cs <- newCapSet(expMethod = 'RAMPAGE', mapped_file = files, sampleNames = fnames) cs <- detectTSS(cs, groups = d$group, outfile_prefix = file.path(outdir, "prefix"), foldChange = 2, ncores = 1)

warnings() Warning messages: 1: In detectTSS(cs, groups = d$group, outfile_prefix = file.path(outdir, ... : Filtered files not found under sampleInfo(CSobject). Using mapped files 2: In mean.default(filter) : argument is not numeric or logical: returning NA 3: In mean.default(filter) : argument is not numeric or logical: returning NA

unique(detected_TSS$score) [1] NA

cs

An object of class CapSet

Experiment method : RAMPAGE FASTQ Read 1 :
FASTQ Read 2 :

Sample information :

DataFrame with 4 rows and 9 columns samples demult_R1 demult_R2 mapped_file filtered_file demult_reads num_mapped num_filtered num_intss

<character> <logical> <logical> <character> <logical> <logical> <integer> <logical> <numeric> 1 4R_siBrf1_Ninfected_marked NA NA ./4R_siBrf1_Ninfected_marked.bam NA NA 38652464 NA 37440001 2 4_siBrf1_Ninfected_marked NA NA ./4_siBrf1_Ninfected_marked.bam NA NA 37277228 NA 35753346 3 6R_siBrf1_Infected_marked NA NA ./6R_siBrf1_Infected_marked.bam NA NA 29026535 NA 28149790 4 6_siBrf1_Infected_marked NA NA ./6_siBrf1_Infected_marked.bam NA NA 36878287 NA 35634222

TSS enrichment information :

Detected TSS per group GRangesList object of length 2: $siBrf1_NI GRanges object with 190191 ranges and 2 metadata columns: seqnames ranges strand | grouping score

<Rle> <IRanges> <Rle> | <ManyToManyGrouping> <numeric> [1] GL000008.2 125621-125630 - | 298150 NA [2] GL000008.2 132806-132820 - | 298151,298152 NA [3] GL000008.2 137126-137140 - | 298153,298154 NA [4] GL000008.2 137191-137200 - | 298155 NA [5] GL000008.2 138681-138695 - | 298156,298157 NA ... ... ... ... . ... ... [190187] chrY 20801321-20801335 - | 586664,586665 NA [190188] chrY 24099216-24099230 - | 586666,586667 NA [190189] chrY 24582311-24582325 - | 586668,586669 NA [190190] chrY 25099881-25099895 - | 586670,586671 NA

[190191] chrY 25099901-25099915 - | 586672,586673 NA

seqinfo: 194 sequences from an unspecified genome; no seqlengths

$siBrf1_I GRanges object with 175260 ranges and 2 metadata columns: seqnames ranges strand | grouping score

<Rle> <IRanges> <Rle> | <ManyToManyGrouping> <numeric> [1] GL000008.2 125591-125605 - | 273806,273807 NA [2] GL000008.2 127041-127055 - | 273808,273809 NA [3] GL000008.2 137126-137145 - | 273810,273811,273812 NA [4] GL000008.2 137201-137225 - | 273813,273814,273815,... NA [5] GL000008.2 140621-140635 - | 273817,273818 NA ... ... ... ... . ... ... [175256] chrY 19744706-19744750 - | 541293,541294,541295,... NA [175257] chrY 26622516-26622530 - | 541300,541301 NA [175258] chrY 56954016-56954030 - | 541302,541303 NA [175259] chrY 56954066-56954080 - | 541304,541305 NA

[175260] chrY 57214471-57214485 - | 541306,541307 NA

seqinfo: 194 sequences from an unspecified genome; no seqlengths

sessionInfo() R version 4.0.1 (2020-06-06) Platform: x86_64-pc-linux-gnu (64-bit) Running under: CentOS Linux 7 (Core)

Matrix products: default BLAS/LAPACK: /usr/lib64/libopenblas-r0.3.3.so

locale: [1] C

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

other attached packages: [1] icetea_1.6.1 colorout_1.2-2

loaded via a namespace (and not attached): [1] Biobase_2.48.0 httr_1.4.2 edgeR_3.30.3 bit64_4.0.5 splines_4.0.1
[6] assertthat_0.2.1 askpass_1.1 stats4_4.0.1 BiocFileCache_1.12.1 latticeExtra_0.6-29
[11] blob_1.2.1 BSgenome_1.56.0 GenomeInfoDbData_1.2.3 Rsamtools_2.4.0 progress_1.2.2
[16] pillar_1.4.6 RSQLite_2.2.1 lattice_0.20-41 limma_3.44.3 glue_1.4.2
[21] digest_0.6.26 GenomicRanges_1.40.0 RColorBrewer_1.1-2 XVector_0.28.0 colorspace_1.4-1
[26] Matrix_1.2-18 DESeq2_1.28.1 XML_3.99-0.5 pkgconfig_2.0.3 ShortRead_1.46.0
[31] csaw_1.22.1 biomaRt_2.44.4 genefilter_1.70.0 zlibbioc_1.34.0 purrr_0.3.4
[36] xtable_1.8-4 scales_1.1.1 jpeg_0.1-8.1 BiocParallel_1.22.0 tibble_3.0.4
[41] openssl_1.4.3 annotate_1.66.0 generics_0.0.2 IRanges_2.22.2 ggplot2_3.3.2
[46] ellipsis_0.3.1 SummarizedExperiment_1.18.2 GenomicFeatures_1.40.1 BiocGenerics_0.34.0 survival_3.2-7
[51] magrittr_1.5 crayon_1.3.4 memoise_1.1.0 hwriter_1.3.2 xml2_1.3.2
[56] tools_4.0.1 prettyunits_1.1.1 hms_0.5.3 lifecycle_0.2.0 matrixStats_0.57.0
[61] stringr_1.4.0 S4Vectors_0.26.1 locfit_1.5-9.4 munsell_0.5.0 DelayedArray_0.14.1
[66] AnnotationDbi_1.50.3 Biostrings_2.56.0 compiler_4.0.1 GenomeInfoDb_1.24.2 rlang_0.4.8
[71] grid_4.0.1 RCurl_1.98-1.2 VariantAnnotation_1.34.0 rappdirs_0.3.1 TxDb.Dmelanogaster.UCSC.dm6.ensGene_3.11.0 [76] bitops_1.0-6 gtable_0.3.0 DBI_1.1.0 curl_4.3 R6_2.4.1
[81] GenomicAlignments_1.24.0 dplyr_1.0.2 rtracklayer_1.48.0 bit_4.0.4 stringi_1.5.3
[86] parallel_4.0.1 Rcpp_1.0.5 png_0.1-7 vctrs_0.3.4 geneplotter_1.66.0
[91] dbplyr_1.4.4 tidyselect_1.1.0