Starting zUMIs

[ISIDORAPA]

Hi, I am new in bioinformatic field and I am trying to do analysis of RNA seq with zUMIs.
I wrote the yaml file according the instruction:

##YAML file- zUMIs.yaml

###########################################
#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: SmartSeq3

#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/isidora/IRCCS_Candiolo/Fastq/_R1_001_val_1.fq ##/home/isidora/IRCCS_Candiolo/Fastq/zUMIsFiles1/ #path to first file
base_definition:
- cDNA(1-150)
- BC(1-8)
- UMI(12-19)
file2:
name: /home/isidora/IRCCS_Candiolo/Fastq/_R2_001_val_2.fq ## /home/isidora/IRCCS_Candiolo/Fastq/zUMIsFiles2/ #path to second file
base_definition:
- cDNA(1-150)
- BC(1-8)
- UMI(12-19)

#reference genome setup
reference:
STAR_index: /home/isidora/IRCCS_Candiolo/Fastq/ #path to STAR genome index
GTF_file: /home/isidora/IRCCS_Candiolo/Fastq/hg38.refGene.gtf #path to gene annotation file in GTF format
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: /home/isidora/IRCCS_Candiolo/Fastq/hg38.fa #Optional parameter. It is possible to give additonal reference sequences here, eg ERCC.fa
additional_STAR_params: pGe.sjdbOverhang > 0 #Optional parameter. you may add custom mapping parameters to STAR here
#output directory
out_dir: /home/isidora/IRCCS_Candiolo/Fastq/zUMIs/ #specify the full path to the output directory

###########################################
#below, you may optionally change default parameters
###########################################

#number of processors to use
num_threads: 20
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: 3
phred: 20
UMI_filter:
num_bases: 2
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: /home/isidora/IRCCS_Candiolo/Fastq/SampleSheet.txt
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 giiven 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: yes #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: 1 #Hamming distance collapsing of UMI sequences.
velocyto: no #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
samtools_exec: samtools #samtools executable
Rscript_exec: Rscript #Rscript executable
STAR_exec: STAR #STAR executable
pigz_exec: pigz #pigz executable

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

When I start zUMIs with zUMIs/zUMIs.sh -c -y ~/IRCCS_Candiolo/Fastq/zUMIs/zUMIs.yaml I get errors:

Using miniconda environment for zUMIs!
note: internal executables will be used instead of those specified in the YAML file!

You provided these parameters:
YAML file: /home/isidora/IRCCS_Candiolo/Fastq/zUMIs/zUMIs.yaml
zUMIs directory: /home/isidora/IRCCS_Candiolo/Fastq/zUMIs
STAR executable STAR
samtools executable samtools
pigz executable pigz
Rscript executable Rscript
RAM limit: null
zUMIs version 2.9.7e

Wed Aug 23 12:06:25 CEST 2023
WARNING: The STAR version used for mapping is 2.7.3a and the STAR index was created using the version 2.7.4a. 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...
sh: 1: Syntax error: Unterminated quoted string
sh: 1: Syntax error: Unterminated quoted string
Wed Aug 23 12:06:26 CEST 2023
Error in eval(bysub, parent.frame(), parent.frame()) :
object 'XC' not found
Calls: cellBC -> [ -> [.data.table -> eval -> eval
In addition: Warning message:
In data.table::fread(bccount_file, header = FALSE, col.names = c("XC", :
File '/home/isidora/IRCCS_Candiolo/Fastq/zUMIs//SmartSeq3.BCstats.txt' has size 0. Returning a NULL data.table.
Execution halted
Mapping...
[1] "2023-08-23 12:06:28 CEST"
Warning message:
In data.table::fread(cmd = paste(samtools, "view", filtered_bams[1], :
File '/tmp/RtmpKSSqU2/file57f5393a8fde' has size 0. Returning a NULL data.table.

EXITING because of fatal PARAMETERS error: pGe.sjdbOverhang <=0 while junctions are inserted on the fly with --sjdbFileChrStartEnd or/and --sjdbGTFfile
SOLUTION: specify pGe.sjdbOverhang>0, ideally readmateLength-1
Aug 23 12:07:18 ...... FATAL ERROR, exiting
Wed Aug 23 12:07:18 CEST 2023
Counting...
[1] "2023-08-23 12:07:30 CEST"
Error in fread(paste0(opt$out_dir, "/zUMIs_output/", opt$project, "kept_barcodes_binned.txt")) :
File '/home/isidora/IRCCS_Candiolo/Fastq/zUMIs//zUMIs_output/SmartSeq3kept_barcodes_binned.txt' does not exist or is non-readable. getwd()=='/home/isidora/IRCCS_Candiolo/Fastq/zUMIs'
Execution halted
Wed Aug 23 12:07:30 CEST 2023
Loading required package: yaml
Loading required package: Matrix
[1] "loomR found"
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/isidora/IRCCS_Candiolo/Fastq/zUMIs//zUMIs_output/expression/SmartSeq3.dgecounts.rds', probable reason 'No such file or directory'
Execution halted
Wed Aug 23 12:07:32 CEST 2023
Descriptive statistics...
[1] "I am loading useful packages for plotting..."
[1] "2023-08-23 12:07:33 CEST"
Error in data.table::fread(paste0(opt$out_dir, "/zUMIs_output/", opt$project, :
File '/home/isidora/IRCCS_Candiolo/Fastq/zUMIs//zUMIs_output/SmartSeq3kept_barcodes.txt' does not exist or is non-readable. getwd()=='/home/isidora/IRCCS_Candiolo/Fastq/zUMIs'
Execution halted
Wed Aug 23 12:07:37 CEST 2023

I do not understand the nature of the errors and what I need to fix, can anyone help me?
Thanks

[cziegenhain]

Hi,

Sorry for the slow reply.
I think there is several issues with your config file, although it is also a bit hard to read here in the GitHub issue.
Please check carefully the reference instructins
https://github.com/sdparekh/zUMIs/wiki/Protocol-specific-setup#smart-seq3

For example, I do not see that you give any fastq input file? just a folder?
You also have a whole genome hg38 fasta file in "additional fasta" section - this is meant for transgenes or things that are NOT part of the regular genome. the full genome must be properly indexed using STAR.
Please check the wiki page more carefully, detailed instructions are available there.

Best,
Christoph