############################################################################## ############################################################################## ### ### Running command: ### ### /home/biocbuild/R/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/R/R/site-library --no-vignettes --timings FLAMES_2.0.2.tar.gz ### ############################################################################## ############################################################################## * using log directory ‘/home/biocbuild/bbs-3.20-bioc/meat/FLAMES.Rcheck’ * using R version 4.4.2 (2024-10-31) * using platform: aarch64-unknown-linux-gnu * R was compiled by aarch64-unknown-linux-gnu-gcc (GCC) 14.2.0 GNU Fortran (GCC) 12.3.1 (openEuler 12.3.1-36.oe2403) * running under: openEuler 24.03 (LTS) * using session charset: UTF-8 * using option ‘--no-vignettes’ * checking for file ‘FLAMES/DESCRIPTION’ ... OK * this is package ‘FLAMES’ version ‘2.0.2’ * package encoding: UTF-8 * checking package namespace information ... OK * checking package dependencies ... OK * checking if this is a source package ... OK * checking if there is a namespace ... OK * checking for hidden files and directories ... OK * checking for portable file names ... OK * checking for sufficient/correct file permissions ... OK * checking whether package ‘FLAMES’ can be installed ... NOTE Found the following notes/warnings: Non-staged installation was used See ‘/home/biocbuild/bbs-3.20-bioc/meat/FLAMES.Rcheck/00install.out’ for details. * used C++ compiler: ‘aarch64-unknown-linux-gnu-g++ (GCC) 14.2.0’ * checking C++ specification ... OK Not all R platforms support C++17 * checking installed package size ... NOTE installed size is 5.2Mb sub-directories of 1Mb or more: data 2.7Mb libs 1.4Mb * checking package directory ... OK * checking ‘build’ directory ... OK * checking DESCRIPTION meta-information ... OK * checking top-level files ... OK * checking for left-over files ... OK * checking index information ... OK * checking package subdirectories ... OK * checking code files for non-ASCII characters ... OK * checking R files for syntax errors ... OK * checking whether the package can be loaded ... OK * checking whether the package can be loaded with stated dependencies ... OK * checking whether the package can be unloaded cleanly ... OK * checking whether the namespace can be loaded with stated dependencies ... OK * checking whether the namespace can be unloaded cleanly ... OK * checking loading without being on the library search path ... OK * checking dependencies in R code ... OK * checking S3 generic/method consistency ... OK * checking replacement functions ... OK * checking foreign function calls ... OK * checking R code for possible problems ... NOTE create_spe: no visible binding for global variable 'barcode' filter_coverage: no visible global function definition for 'starts_with' filter_coverage: no visible binding for global variable 'filter_res' find_barcode: no visible binding for global variable 'Sample' find_barcode: no visible binding for global variable 'Outfile' find_variants_grange: no visible binding for global variable 'which_label' find_variants_grange: no visible binding for global variable 'nucleotide' find_variants_grange: no visible binding for global variable 'pos' find_variants_grange: no visible binding for global variable 'count' find_variants_grange: no visible binding for global variable 'counts_no_ins' find_variants_grange: no visible binding for global variable 'ref' generate_sc_sce: no visible binding for global variable 'FSM_match' get_coverage: no visible binding for global variable 'Freq' homopolymer_pct : : no visible binding for global variable 'Freq' homopolymer_pct : : no visible binding for global variable 'pct' plot_coverage: no visible binding for global variable 'tr_length' plot_coverage: no visible binding for global variable 'read_counts' plot_coverage: no visible binding for global variable 'total_counts' plot_coverage: no visible binding for global variable 'cumpct' plot_coverage: no visible binding for global variable 'length_bin' plot_coverage: no visible binding for global variable 'min_length' plot_coverage: no visible binding for global variable 'max_length' plot_coverage: no visible global function definition for 'head' plot_coverage: no visible binding for global variable 'transcript' plot_demultiplex: no visible binding for global variable 'CellBarcode' plot_demultiplex: no visible binding for global variable 'Sample' plot_demultiplex: no visible binding for global variable 'UMI' plot_demultiplex: no visible binding for global variable 'UMI_count' plot_demultiplex: no visible binding for global variable 'barcode_rank' plot_demultiplex: no visible binding for global variable 'FlankEditDist' plot_demultiplex: no visible binding for global variable 'n_reads' plot_demultiplex: no visible binding for global variable 'BarcodeEditDist' plot_demultiplex: no visible binding for global variable 'total reads' plot_demultiplex: no visible binding for global variable 'demultiplexed reads' plot_demultiplex: no visible binding for global variable 'single match reads' plot_demultiplex: no visible binding for global variable 'undemultiplexted reads' plot_demultiplex: no visible binding for global variable 'multi-matching reads' plot_demultiplex: no visible binding for global variable 'Type' plot_demultiplex: no visible binding for global variable 'Reads' plot_demultiplex: no visible binding for global variable 'input' plot_demultiplex: no visible binding for global variable 'output' plot_demultiplex: no visible binding for global variable 'read1_with_adapter' plot_demultiplex: no visible binding for global variable 'Count' plot_flagstat: no visible global function definition for 'everything' plot_flagstat: no visible binding for global variable 'name' plot_flagstat: no visible binding for global variable 'value' plot_isoform_heatmap : group_annotation: no visible global function definition for 'HeatmapAnnotation' plot_isoform_reduced_dim: no visible binding for global variable 'x' plot_isoform_reduced_dim: no visible binding for global variable 'y' plot_isoform_reduced_dim: no visible binding for global variable 'expr' plot_spatial_isoform: no visible global function definition for 'head' plot_spatial_pie: no visible global function definition for 'head' plot_spatial_pie: no visible binding for global variable 'imageY' plot_spatial_pie: no visible binding for global variable 'imageX' relative_mutation_positions: no visible binding for global variable 'region' relative_mutation_positions_single: no visible binding for global variable 'type' sc_DTU_analysis: no visible binding for global variable 'FSM_match' sc_DTU_analysis: no visible binding for global variable 'gene_id' sc_DTU_analysis: no visible binding for global variable '.' sc_DTU_analysis: no visible binding for global variable 'cell_id' sc_DTU_analysis: no visible binding for global variable 'cnt' sc_DTU_analysis: no visible binding for global variable 'tr_id' sc_DTU_analysis: no visible binding for global variable 'label' sc_DTU_analysis : filter_tr: no visible binding for global variable 'gene_id' sc_DTU_analysis : filter_tr: no visible binding for global variable '.' sc_mutations: no visible binding for global variable 'mutation_index' sc_mutations: no visible binding for global variable 'bam_index' variant_count_tb: no visible binding for global variable 'barcode' variant_count_tb: no visible binding for global variable 'allele_count' variant_count_tb: no visible binding for global variable 'cell_total_reads' Undefined global functions or variables: . BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq HeatmapAnnotation Outfile Reads Sample Type UMI UMI_count allele_count bam_index barcode barcode_rank cell_id cell_total_reads cnt count counts_no_ins cumpct demultiplexed reads everything expr filter_res gene_id head imageX imageY input label length_bin max_length min_length multi-matching reads mutation_index n_reads name nucleotide output pct pos read1_with_adapter read_counts ref region single match reads starts_with total reads total_counts tr_id tr_length transcript type undemultiplexted reads value which_label x y Consider adding importFrom("base", "match", "single") importFrom("utils", "head") to your NAMESPACE file. * checking Rd files ... OK * checking Rd metadata ... OK * checking Rd cross-references ... OK * checking for missing documentation entries ... OK * checking for code/documentation mismatches ... OK * checking Rd \usage sections ... OK * checking Rd contents ... OK * checking for unstated dependencies in examples ... OK * checking contents of ‘data’ directory ... OK * checking data for non-ASCII characters ... OK * checking LazyData ... OK * checking data for ASCII and uncompressed saves ... OK * checking line endings in shell scripts ... OK * checking line endings in C/C++/Fortran sources/headers ... OK * checking line endings in Makefiles ... OK * checking compilation flags in Makevars ... OK * checking for GNU extensions in Makefiles ... NOTE GNU make is a SystemRequirements. * checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK * checking use of PKG_*FLAGS in Makefiles ... OK * checking compiled code ... NOTE Note: information on .o files is not available File ‘/home/biocbuild/R/R-4.4.2/site-library/FLAMES/libs/FLAMES.so’: Found ‘abort’, possibly from ‘abort’ (C) Found ‘exit’, possibly from ‘exit’ (C) Found ‘stderr’, possibly from ‘stderr’ (C) Found ‘stdout’, possibly from ‘stdout’ (C) Compiled code should not call entry points which might terminate R nor write to stdout/stderr instead of to the console, nor use Fortran I/O nor system RNGs nor [v]sprintf. The detected symbols are linked into the code but might come from libraries and not actually be called. See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual. * checking files in ‘vignettes’ ... OK * checking examples ... ERROR Running examples in ‘FLAMES-Ex.R’ failed The error most likely occurred in: > base::assign(".ptime", proc.time(), pos = "CheckExEnv") > ### Name: bulk_long_pipeline > ### Title: Pipeline for Bulk Data > ### Aliases: bulk_long_pipeline > > ### ** Examples > > # download the two fastq files, move them to a folder to be merged together > temp_path <- tempfile() > bfc <- BiocFileCache::BiocFileCache(temp_path, ask = FALSE) > file_url <- + "https://raw.githubusercontent.com/OliverVoogd/FLAMESData/master/data" > # download the required fastq files, and move them to new folder > fastq1 <- bfc[[names(BiocFileCache::bfcadd(bfc, "Fastq1", paste(file_url, "fastq/sample1.fastq.gz", sep = "/")))]] > fastq2 <- bfc[[names(BiocFileCache::bfcadd(bfc, "Fastq2", paste(file_url, "fastq/sample2.fastq.gz", sep = "/")))]] > annotation <- bfc[[names(BiocFileCache::bfcadd(bfc, "annot.gtf", paste(file_url, "SIRV_isoforms_multi-fasta-annotation_C_170612a.gtf", sep = "/")))]] > genome_fa <- bfc[[names(BiocFileCache::bfcadd(bfc, "genome.fa", paste(file_url, "SIRV_isoforms_multi-fasta_170612a.fasta", sep = "/")))]] > fastq_dir <- paste(temp_path, "fastq_dir", sep = "/") # the downloaded fastq files need to be in a directory to be merged together > dir.create(fastq_dir) > file.copy(c(fastq1, fastq2), fastq_dir) [1] TRUE TRUE > unlink(c(fastq1, fastq2)) # the original files can be deleted > > outdir <- tempfile() > dir.create(outdir) > se <- bulk_long_pipeline( + annotation = annotation, fastq = fastq_dir, outdir = outdir, genome_fa = genome_fa, + config_file = create_config(outdir, type = "sc_3end", threads = 1, no_flank = TRUE) + ) Writing configuration parameters to: /home/biocbuild/tmp/RtmpWjzrGa/file2805747fc88735/config_file_2622836.json #### Input parameters: { "pipeline_parameters": { "seed": [2022], "threads": [1], "do_barcode_demultiplex": [true], "do_gene_quantification": [true], "do_genome_alignment": [true], "do_isoform_identification": [true], "bambu_isoform_identification": [false], "multithread_isoform_identification": [false], "do_read_realignment": [true], "do_transcript_quantification": [true], "oarfish_quantification": [true] }, "barcode_parameters": { "max_bc_editdistance": [2], "max_flank_editdistance": [8], "pattern": { "primer": ["CTACACGACGCTCTTCCGATCT"], "BC": ["NNNNNNNNNNNNNNNN"], "UMI": ["NNNNNNNNNNNN"], "polyT": ["TTTTTTTTT"] }, "strand": ["-"], "TSO_seq": ["AAGCAGTGGTATCAACGCAGAGTACATGGG"], "TSO_prime": [3], "full_length_only": [false] }, "isoform_parameters": { "generate_raw_isoform": [false], "max_dist": [10], "max_ts_dist": [100], "max_splice_match_dist": [10], "min_fl_exon_len": [40], "max_site_per_splice": [3], "min_sup_cnt": [5], "min_cnt_pct": [0.001], "min_sup_pct": [0.2], "bambu_trust_reference": [true], "strand_specific": [0], "remove_incomp_reads": [4], "downsample_ratio": [1] }, "alignment_parameters": { "use_junctions": [true], "no_flank": [true] }, "realign_parameters": { "use_annotation": [true] }, "transcript_counting": { "min_tr_coverage": [0.4], "min_read_coverage": [0.4] } } gene annotation: /home/biocbuild/tmp/RtmpWjzrGa/file2805746992a4bb/280574414041f9_SIRV_isoforms_multi-fasta-annotation_C_170612a.gtf genome fasta: /home/biocbuild/tmp/RtmpWjzrGa/file2805746992a4bb/2805744cad85b4_SIRV_isoforms_multi-fasta_170612a.fasta input fastq files: /home/biocbuild/tmp/RtmpWjzrGa/file2805746992a4bb/fastq_dir/2805743185c986_sample2.fastq.gz /home/biocbuild/tmp/RtmpWjzrGa/file2805746992a4bb/fastq_dir/2805743623ff42_sample1.fastq.gz output directory: /home/biocbuild/tmp/RtmpWjzrGa/file2805747fc88735 minimap2 path: k8 path: #### Aligning reads to genome using minimap2 Aligning sample 2805743185c986_sample2 ... 07:09:58 Tue Jan 21 2025 minimap2_align [M::mm_idx_gen::0.013*0.96] collected minimizers [M::mm_idx_gen::0.022*0.97] sorted minimizers [M::main::0.022*0.97] loaded/built the index for 7 target sequence(s) [M::mm_mapopt_update::0.024*0.98] mid_occ = 14 [M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 7 [M::mm_idx_stat::0.025*0.98] distinct minimizers: 39103 (61.08% are singletons); average occurrences: 1.935; average spacing: 2.947; total length: 223019 [M::worker_pipeline::8.466*0.99] mapped 2500 sequences [M::main] Version: 2.24-r1122 [M::main] CMD: /usr/bin/minimap2 -ax splice -t 1 -k14 --secondary=no --seed 2022 --splice-flank=no --junc-bed /home/biocbuild/tmp/RtmpWjzrGa/file2805747fc88735/tmp_splice_anno.bed12 --junc-bonus 1 /home/biocbuild/tmp/RtmpWjzrGa/file2805746992a4bb/2805744cad85b4_SIRV_isoforms_multi-fasta_170612a.fasta /home/biocbuild/tmp/RtmpWjzrGa/file2805746992a4bb/fastq_dir/2805743185c986_sample2.fastq.gz [M::main] Real time: 8.470 sec; CPU: 8.367 sec; Peak RSS: 0.111 GB Aligning sample 2805743623ff42_sample1 ... 07:10:07 Tue Jan 21 2025 minimap2_align [M::mm_idx_gen::0.014*1.03] collected minimizers [M::mm_idx_gen::0.024*1.02] sorted minimizers [M::main::0.024*1.02] loaded/built the index for 7 target sequence(s) [M::mm_mapopt_update::0.026*1.02] mid_occ = 14 [M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 7 [M::mm_idx_stat::0.028*1.01] distinct minimizers: 39103 (61.08% are singletons); average occurrences: 1.935; average spacing: 2.947; total length: 223019 [M::worker_pipeline::8.479*0.99] mapped 2500 sequences [M::main] Version: 2.24-r1122 [M::main] CMD: /usr/bin/minimap2 -ax splice -t 1 -k14 --secondary=no --seed 2022 --splice-flank=no --junc-bed /home/biocbuild/tmp/RtmpWjzrGa/file2805747fc88735/tmp_splice_anno.bed12 --junc-bonus 1 /home/biocbuild/tmp/RtmpWjzrGa/file2805746992a4bb/2805744cad85b4_SIRV_isoforms_multi-fasta_170612a.fasta /home/biocbuild/tmp/RtmpWjzrGa/file2805746992a4bb/fastq_dir/2805743623ff42_sample1.fastq.gz [M::main] Real time: 8.484 sec; CPU: 8.360 sec; Peak RSS: 0.081 GB 07:10:15 Tue Jan 21 2025 find_isoform #### Read gene annotations Removed similar transcripts in gene annotation: Counter({'duplicated_transcripts': 2}) #### find isoforms SIRV1 SIRV2 SIRV3 SIRV4 SIRV5 SIRV6 SIRV7 #### Realign to transcript using minimap2 Realigning sample 2805743185c986_sample2 ... 07:10:18 Tue Jan 21 2025 minimap2_realign [M::mm_idx_gen::0.004*1.19] collected minimizers [M::mm_idx_gen::0.007*1.71] sorted minimizers [M::main::0.007*1.70] loaded/built the index for 84 target sequence(s) [M::mm_mapopt_update::0.007*1.66] mid_occ = 19 [M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 84 [M::mm_idx_stat::0.008*1.63] distinct minimizers: 4795 (32.30% are singletons); average occurrences: 3.260; average spacing: 5.403; total length: 84456 [M::worker_pipeline::0.909*2.48] mapped 2500 sequences [M::main] Version: 2.24-r1122 [M::main] CMD: /usr/bin/minimap2 --eqx -N 100 -ax map-ont /home/biocbuild/tmp/RtmpWjzrGa/file2805747fc88735/transcript_assembly.fa /home/biocbuild/tmp/RtmpWjzrGa/file2805746992a4bb/fastq_dir/2805743185c986_sample2.fastq.gz [M::main] Real time: 0.911 sec; CPU: 2.254 sec; Peak RSS: 0.027 GB file renamed to 2805743185c986_sample2_realign2transcript.bam Warning in file.remove(file.path(outdir, paste0(prefix, "tmp_align.bam"))) : cannot remove file '/home/biocbuild/tmp/RtmpWjzrGa/file2805747fc88735/2805743185c986_sample2_tmp_align.bam', reason 'No such file or directory' Realigning sample 2805743623ff42_sample1 ... 07:10:19 Tue Jan 21 2025 minimap2_realign [M::mm_idx_gen::0.004*0.97] collected minimizers [M::mm_idx_gen::0.007*1.57] sorted minimizers [M::main::0.007*1.57] loaded/built the index for 84 target sequence(s) [M::mm_mapopt_update::0.008*1.53] mid_occ = 19 [M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 84 [M::mm_idx_stat::0.008*1.51] distinct minimizers: 4795 (32.30% are singletons); average occurrences: 3.260; average spacing: 5.403; total length: 84456 [M::worker_pipeline::1.238*2.50] mapped 2500 sequences [M::main] Version: 2.24-r1122 [M::main] CMD: /usr/bin/minimap2 --eqx -N 100 -ax map-ont /home/biocbuild/tmp/RtmpWjzrGa/file2805747fc88735/transcript_assembly.fa /home/biocbuild/tmp/RtmpWjzrGa/file2805746992a4bb/fastq_dir/2805743623ff42_sample1.fastq.gz [M::main] Real time: 1.240 sec; CPU: 3.101 sec; Peak RSS: 0.032 GB file renamed to 2805743623ff42_sample1_realign2transcript.bam Warning in file.remove(file.path(outdir, paste0(prefix, "tmp_align.bam"))) : cannot remove file '/home/biocbuild/tmp/RtmpWjzrGa/file2805747fc88735/2805743623ff42_sample1_tmp_align.bam', reason 'No such file or directory' #### Generating transcript count matrix [2m2025-01-21T07:10:21.168727Z[0m [32m INFO[0m [2moarfish[0m[2m:[0m setting user-provided filter parameters. [2m2025-01-21T07:10:21.170878Z[0m [32m INFO[0m [2moarfish::alignment_parser[0m[2m:[0m read header from BAM file /home/biocbuild/tmp/RtmpWjzrGa/file2805747fc88735/2805743185c986_sample2_realign2transcript.bam, contains 84 reference sequences. thread 'main' panicked at src/alignment_parser.rs:29:10: has inner header note: run with `RUST_BACKTRACE=1` environment variable to display a backtrace Error in run_oarfish(realign_bam, outdir, threads = config$pipeline_parameters$threads, : error running oarfish: 134 Calls: bulk_long_pipeline ... quantify_transcript -> quantify_transcript_oarfish -> run_oarfish Execution halted * checking for unstated dependencies in ‘tests’ ... OK * checking tests ... Running ‘testthat.R’ OK * checking for unstated dependencies in vignettes ... OK * checking package vignettes ... OK * checking running R code from vignettes ... SKIPPED * checking re-building of vignette outputs ... SKIPPED * checking PDF version of manual ... OK * DONE Status: 1 ERROR, 5 NOTEs See ‘/home/biocbuild/bbs-3.20-bioc/meat/FLAMES.Rcheck/00check.log’ for details.