search

sdparekh / zUMIs

zUMIs: A fast and flexible pipeline to process RNA sequencing data with UMIs
GNU General Public License v3.0
275 stars 67 forks source link

Error running zUMIs #72

Closed ghoshal closed 6 years ago

ghoshal commented 6 years ago

Hi,

I am getting an error while running zUMIs on split seq data. The program gets stuck at this particular step.

bash zUMIs-master.sh -y /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/zUMIs_0078.yaml

You provided these parameters: YAML file: /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/zUMIs_0078.yaml zUMIs directory: /Users/biba/Downloads/softwares/zUMIs STAR executable STAR samtools executable samtools pigz executable pigz Rscript executable Rscript RAM limit: null zUMIs version 2.0.6

Filtering... split: illegal option -- - usage: split [-a sufflen] [-b byte_count] [-l line_count] [-p pattern] [file [prefix]] split: illegal option -- - usage: split [-a sufflen] [-b byte_count] [-l line_count] [-p pattern] [file [prefix]] /Users/biba/Downloads/softwares/zUMIs/splitfq.sh: line 41: /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/sample_0078/zUMIs_output/.tmpMerge///Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/BC_0078_R1.fastq.gz.listPrefix.txt: No such file or directory /Users/biba/Downloads/softwares/zUMIs/splitfq.sh: line 41: /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/sample_0078/zUMIs_output/.tmpMerge///Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/BC_0078_R1.fastq.gz.listPrefix.txt: No such file or directory ls: /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/sample_0078/zUMIs_output/.tmpMerge/BC_0078_R1.fastq: No such file or directory ls: /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/sample_0078/zUMIs_output/.tmpMerge/BC_0078_R1.fastq: No such file or directory ls: /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/sample_0078/zUMIs_output/.tmpMerge/BC_0078_R1.fastq*: No such file or directory /Users/biba/Downloads/softwares/zUMIs/mergeBAM.sh: line 14: /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/sample_0078/zUMIs_output/.tmpMerge///Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/sample_0078.bamlist.txt: No such file or directory /Users/biba/Downloads/softwares/zUMIs/mergeBAM.sh: line 17: /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/zUMIs_0078.yaml//Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/sample_0078.BCstats.txt: Not a directory head: /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/sample_0078/zUMIs_output/.tmpMerge///Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/sample_0078.bamlist.txt: No such file or directory

Here is my YAML file.

###########################################

Welcome to zUMIs

below, please fill the mandatory inputs

We expect full paths for all files.

###########################################

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

project:sample_0078

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: /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/BC_0078_R1.fastq.gz base_definition:

reference genome setup

reference: STAR_index: /Users/biba/Downloads/softwares/mm10/star_indices GTF_file: /Users/biba/Downloads/softwares/mm10/annotation/Mus_musculus.GRCm38.77.gtf 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: /Volumes/Promise_Pegasus_New/Single_cell_seq/September_2018/Seda/samples/sample_0078

###########################################

below, you may optionally change default parameters

###########################################

number of processors to use

num_threads: 16 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: 2 phred: 10 UMI_filter: num_bases: 2 phred: 10

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 BarcodeBinning: 0 #Hamming distance binning of close cell barcode sequences. ATTENTION! This option is currently not implemented! nReadsperCell: 50 #Keep only the cell barcodes with atleast n number of reads.

Options related to counting of reads towards expression profiles

counting_opts: introns: yes #can be set to no for exon-only counting. 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: no #Would you like velocyto-compatible counting of intron-exon spanning reads primaryHit: yes #Do you want to count the primary Hits of multimapping reads towards gene expression levels? 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

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

samtools_exec: samtools pigz_exec: pigz Rscript_exec: Rscript STAR_exec: STAR

zUMIs_directory: /Users/biba/Downloads/softwares/zUMIs

Any help would be awesome.

Thanks,

Bibaswan

cziegenhain commented 6 years ago

Hey Bibaswan,

This looks like issue #70. What OS are you trying to run this on?

ghoshal commented 6 years ago

Hi,

I am running the program on Mac OSX Mojave.

Thanks,

Bibaswan

On Thu, Sep 20, 2018, 1:26 AM cziegenhain, notifications@github.com wrote:

Hey Bibaswan,

This looks like issue #70 https://github.com/sdparekh/zUMIs/issues/70. What OS are you trying to run this on?

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/sdparekh/zUMIs/issues/72#issuecomment-423044385, or mute the thread https://github.com/notifications/unsubscribe-auth/AKyzlf429J24Rapm_LM3S0PmLF1FYD7Nks5ucycXgaJpZM4WxA7r .

cziegenhain commented 6 years ago

Hey Bibaswan,

sorry, Mac OS is neither tested nor supported by zUMIs. I guess that here, the Mac OS split command lacks the necessary functionalities. Maybe a EC2 instance on AWS could be helpful for you?

Best, Christoph

ghoshal commented 6 years ago

Hi Christoph,

I will try this in a local cluster environment and see.

Thanks,

Bibaswan

cziegenhain commented 6 years ago

Excellent, let us know if you encounter further issues.