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sdparekh / zUMIs

zUMIs: A fast and flexible pipeline to process RNA sequencing data with UMIs
GNU General Public License v3.0
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BUG: split cannot open path error #404

Open YinCY123 opened 1 month ago

YinCY123 commented 1 month ago

Describe the bug during filtering step the split: cannot open path error consist occur.

To Reproduce ###########################################

Welcome to zUMIs

below, please fill the mandatory inputs

We expect full paths for all files.

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define a project name that will be used to name output files

project: GSE129798

Sequencing File Inputs:

For each input file, make one list object & define path and barcode ranges

base definition vocabulary: BC(n) UMI(n) cDNA(n).

Barcode range definition needs to account for all ranges. You can give several comma-separated ranges for BC & UMI sequences, eg. BC(1-6,20-26)

you can specify between 1 and 4 input files

sequence_files: file1: name: /home/yincy/git/disk14/kidney/GSE129798/bam/Mouse_adult_Female1_zone1.bam base_definition:

reference genome setup

reference: STAR_index: /home/yincy/git/data/biodata/index/mouse/star_mm10_index/ GTF_file: /home/yincy/git/data/biodata/annotation/mouse/gencode.vM23.primary_assembly.annotation.gtf exon_extension: no #extend exons by a certain width? extension_length: 0 #number of bp to extend exons by scaffold_length_min: 0 #minimal scaffold/chromosome length to consider (0 = all) additional_files: #Optional parameter. It is possible to give additional reference sequences here, eg ERCC.fa additional_STAR_params: #Optional parameter. you may add custom mapping parameters to STAR here

output directory

out_dir: /home/yincy/git/zUMIs

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below, you may optionally change default parameters

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number of processors to use

num_threads: 10 mem_limit: null #Memory limit in Gigabytes, null meaning unlimited RAM usage.

barcode & UMI filtering options

number of bases under the base quality cutoff that should be filtered out.

Phred score base-cutoff for quality control.

filter_cutoffs: BC_filter: num_bases: 1 phred: 20 UMI_filter: num_bases: 1 phred: 20

Options for Barcode handling

You can give either number of top barcodes to use or give an annotation of cell barcodes.

If you leave both barcode_num and barcode_file empty, zUMIs will perform automatic cell barcode selection for you!

barcodes: barcode_num: null barcode_file: null barcode_sharing: null #Optional for combining several barcode sequences per cell (see github wiki) automatic: yes #Give yes/no to this option. If the cell barcodes should be detected automatically. If the barcode file is given in combination with automatic barcode detection, the list of given barcodes will be used as whitelist. BarcodeBinning: 1 #Hamming distance binning of close cell barcode sequences. nReadsperCell: 100 #Keep only the cell barcodes with atleast n number of reads. demultiplex: no #produce per-cell demultiplexed bam files.

Options related to counting of reads towards expression profiles

counting_opts: introns: yes #can be set to no for exon-only counting. intronProb: no #perform an estimation of how likely intronic reads are to be derived from mRNA by comparing to intergenic counts. downsampling: 0 #Number of reads to downsample to. This value can be a fixed number of reads (e.g. 10000) or a desired range (e.g. 10000-20000) Barcodes with less than will not be reported. 0 means adaptive downsampling. Default: 0. strand: 0 #Is the library stranded? 0 = unstranded, 1 = positively stranded, 2 = negatively stranded Ham_Dist: 0 #Hamming distance collapsing of UMI sequences. velocyto: yes #Would you like velocyto to do counting of intron-exon spanning reads primaryHit: yes #Do you want to count the primary Hits of multimapping reads towards gene expression levels? multi_overlap: no #Do you want to assign reads overlapping to multiple features? fraction_overlap: 0 #minimum required fraction of the read overlapping with the gene for read assignment to genes twoPass: yes #perform basic STAR twoPass mapping

produce stats files and plots?

make_stats: yes

Start zUMIs from stage. Possible TEXT(Filtering, Mapping, Counting, Summarising). Default: Filtering.

which_Stage: Filtering

define dependencies program paths

below, fqfilter will add a read_layout flag defining SE or PE

samtools_exec: samtools pigz_exec: pigz STAR_exec: STAR Rscript_exec: Rscript zUMIs_directory: /home/yincy/tools/zUMIs read_layout: PE

Screenshots

Good news! A newer version of zUMIs is available at https://github.com/sdparekh/zUMIs

Using miniconda environment for zUMIs! note: internal executables will be used instead of those specified in the YAML file! Warning message: package ‘yaml’ was built under R version 4.4.1

You provided these parameters: YAML file: git/kidney/bash/zUMIs_GSE129798.run.yaml zUMIs directory: /home/yincy/tools/zUMIs STAR executable STAR samtools executable samtools pigz executable pigz Rscript executable Rscript RAM limit: null zUMIs version 2.9.7e

Mon Oct 7 07:46:08 PM CST 2024 WARNING: The STAR version used for mapping is 2.7.3a and the STAR index was created using the version 20201. This may lead to an error while mapping. If you encounter any errors at the mapping stage, please make sure to create the STAR index using STAR 2.7.3a. Filtering... split: cannot open '#path' for reading: No such file or directory ls: cannot access '/home/yincy/git/zUMIs/zUMIs_output/.tmpMerge/#pathGSE129798': No such file or directory Warning message: package ‘yaml’ was built under R version 4.4.1 Warning message: package ‘yaml’ was built under R version 4.4.1 Warning message: package ‘yaml’ was built under R version 4.4.1 Warning message: package ‘yaml’ was built under R version 4.4.1 Warning message: package ‘yaml’ was built under R version 4.4.1 Warning message: package ‘yaml’ was built under R version 4.4.1 Warning message: package ‘yaml’ was built under R version 4.4.1 couldn't open WARNING: for reading: No such file or directory couldn't open WARNING: for reading: No such file or directory couldn't open ignoring for reading: No such file or directory couldn't open ignoring for reading: No such file or directory couldn't open environment for reading: No such file or directory couldn't open environment for reading: No such file or directory couldn't open value for reading: No such file or directory couldn't open value for reading: No such file or directory couldn't open of for reading: No such file or directory couldn't open of for reading: No such file or directory couldn't open R_HOME for reading: No such file or directory couldn't open R_HOME for reading: No such file or directory Warning message: package ‘yaml’ was built under R version 4.4.1 sh: 1: NULL: not found sh: 1: NULL: not found couldn't open WARNING: for reading: No such file or directory couldn't open ignoring for reading: No such file or directory couldn't open environment for reading: No such file or directory couldn't open value for reading: No such file or directory couldn't open of for reading: No such file or directory couldn't open R_HOME for reading: No such file or directory Warning message: package ‘yaml’ was built under R version 4.4.1 couldn't open WARNING: for reading: No such file or directory couldn't open ignoring for reading: No such file or directory couldn't open environment for reading: No such file or directory couldn't open value for reading: No such file or directory couldn't open of for reading: No such file or directory couldn't open R_HOME for reading: No such file or directory couldn't open WARNING: for reading: No such file or directory couldn't open ignoring for reading: No such file or directory couldn't open environment for reading: No such file or directory couldn't open value for reading: No such file or directory couldn't open of for reading: No such file or directory couldn't open R_HOME for reading: No such file or directory couldn't open WARNING: for reading: No such file or directory couldn't open ignoring for reading: No such file or directory couldn't open environment for reading: No such file or directory couldn't open value for reading: No such file or directory couldn't open of for reading: No such file or directory couldn't open R_HOME for reading: No such file or directory sh: 1: NULL: not found sh: 1: NULL: not found sh: 1: NULL: not found sh: 1: NULL: not found couldn't open WARNING: for reading: No such file or directory couldn't open ignoring for reading: No such file or directory couldn't open environment for reading: No such file or directory couldn't open value for reading: No such file or directory couldn't open of for reading: No such file or directory couldn't open R_HOME for reading: No such file or directory sh: 1: NULL: not found couldn't open WARNING: for reading: No such file or directory couldn't open ignoring for reading: No such file or directory couldn't open environment for reading: No such file or directory couldn't open value for reading: No such file or directory couldn't open of for reading: No such file or directory couldn't open R_HOME for reading: No such file or directory sh: 1: NULL: not found couldn't open WARNING: for reading: No such file or directory couldn't open ignoring for reading: No such file or directory couldn't open environment for reading: No such file or directory couldn't open value for reading: No such file or directory couldn't open of for reading: No such file or directory couldn't open R_HOME for reading: No such file or directory sh: 1: NULL: not found ls: cannot access '/home/yincy/git/zUMIs/zUMIs_output/.tmpMerge//GSE129798..filtered.tagged.bam': No such file or directory cat: '/home/yincy/git/zUMIs/zUMIs_output/.tmpMerge//GSE129798..BCstats.txt': No such file or directory rm: cannot remove '/home/yincy/git/zUMIs/zUMIs_output/.tmpMerge/#pathgz': No such file or directory Mon Oct 7 07:48:00 PM CST 2024 WARNING: ignoring environment value of R_HOME Error: package or namespace load failed for ‘data.table’: .onLoad failed in loadNamespace() for 'data.table', details: call: fun(libname, pkgname) error: This is R 3.6.3 but data.table has been installed using R 4.4.1. The major version must match. Please reinstall data.table. In addition: Warning message: package ‘data.table’ was built under R version 4.4.1 Execution halted Mapping... WARNING: ignoring environment value of R_HOME Warning message: package ‘yaml’ was built under R version 4.4.1 Warning message: package ‘data.table’ was built under R version 4.4.1 [1] "2024-10-07 19:48:00 CST" Error: .onLoad failed in loadNamespace() for 'data.table', details: call: fun(libname, pkgname) error: This is R 3.6.3 but data.table has been installed using R 4.4.1. The major version must match. Please reinstall data.table. Execution halted Mon Oct 7 07:48:07 PM CST 2024 Counting... WARNING: ignoring environment value of R_HOME Error: package or namespace load failed for ‘data.table’: .onLoad failed in loadNamespace() for 'data.table', details: call: fun(libname, pkgname) error: This is R 3.6.3 but data.table has been installed using R 4.4.1. The major version must match. Please reinstall data.table. In addition: Warning message: package ‘data.table’ was built under R version 4.4.1 Execution halted Mon Oct 7 07:48:07 PM CST 2024 WARNING: ignoring environment value of R_HOME Loading required package: yaml Warning message: package ‘yaml’ was built under R version 4.4.1 Loading required package: Matrix Failed with error: ‘This is R 3.6.3, package ‘Matrix’ needs >= 4.4.0’ [1] "loomR found" Warning messages: 1: package ‘loomR’ was built under R version 4.4.1 2: package ‘R6’ was built under R version 4.4.1 3: package ‘hdf5r’ was built under R version 4.4.1 4: package ‘iterators’ was built under R version 4.4.1 5: package ‘itertools’ was built under R version 4.4.1 Error in gzfile(file, "rb") : cannot open the connection Calls: rds_to_loom -> readRDS -> gzfile In addition: Warning message: In gzfile(file, "rb") : cannot open compressed file '/home/yincy/git/zUMIs/zUMIs_output/expression/GSE129798.dgecounts.rds', probable reason 'No such file or directory' Execution halted Mon Oct 7 07:48:08 PM CST 2024 WARNING: ignoring environment value of R_HOME Warning message: package ‘yaml’ was built under R version 4.4.1 [1] "2024-10-07 19:48:08 CST" [1] "Preparing bam file for velocyto..." [E::hts_open_format] Failed to open file NA samtools view: failed to open "NA" for reading: No such file or directory [1] "2024-10-07 19:48:08 CST" Error: .onLoad failed in loadNamespace() for 'data.table', details: call: fun(libname, pkgname) error: This is R 3.6.3 but data.table has been installed using R 4.4.1. The major version must match. Please reinstall data.table. Execution halted Descriptive statistics... WARNING: ignoring environment value of R_HOME [1] "I am loading useful packages for plotting..." [1] "2024-10-07 19:48:08 CST" Error: package or namespace load failed for ‘data.table’: .onLoad failed in loadNamespace() for 'data.table', details: call: fun(libname, pkgname) error: This is R 3.6.3 but data.table has been installed using R 4.4.1. The major version must match. Please reinstall data.table. In addition: Warning message: package ‘data.table’ was built under R version 4.4.1 Execution halted Mon Oct 7 07:48:08 PM CST 2024

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YinCY123 commented 1 month ago

Hi @swatidparekh, can you help fix this bug?