Unable to get past "Export of the viral SAM file done for XXX"

[14zac2]

Hello!

I'm trying to use Viral-Track to analyze single-cell woodchuck liver samples infected with the woodchuck hepatitis virus. When running your program, I experience the same error as mentioned #4 . Although R seemed to recognize the %dopar% command when running through the code line-by-line, that step was consistently throwing giving the error Error in unserialize(socklist[[n]]) : error reading from connection. As you recommended in the thread, I tried changing both parallel loops involving %dopar% to regular for loops, but now I am experiencing a new error:

Loading of the libraries.... ... done ! Warning message:
Output directory does not exist ! Creating it !
1 Fastq files are going to be processed !
Mapping L207_TLH_S7_R2_extracted.fastq file
Apr 04 04:43:31 ..... started STAR run
Apr 04 04:43:32 ..... loading genome
Apr 04 05:10:34 ..... started 1st pass mapping
Apr 04 05:25:23 ..... finished 1st pass mapping
Apr 04 05:25:24 ..... inserting junctions into the genome indices
Apr 04 05:27:40 ..... started mapping
Apr 04 06:29:33 ..... finished mapping
Apr 04 06:29:34 ..... started sorting BAM
Apr 04 06:36:05 ..... finished successfully
Mapping ofL207_TLH_S7_R2_extracted.fastq done !
All fastq files have been mapped successfully
Starting the BAM file analysis
Indexing of the bam file for L207_TLH_S7_R2_extracted is done
Computing stat file for the bam file for L207_TLH_S7_R2_extracted is done
Checking the mapping quality of each virus...
Export of the viral SAM file done for L207_TLH_S7_R2_extracted
Error in h(simpleError(msg, call)) :
  error in evaluating the argument 'flesh' in selecting a method for function 'relist': XStringSet object is too big to be unlisted (would result in an XString
  object of length 2^31 or more)
Calls: as ... unlist -> unlist -> .Call2 -> .handleSimpleError -> h
In addition: There were 50 or more warnings (use warnings() to see the first 50)
Execution halted

Do you have any idea as to why this might be occurring? I am worried that it might be due to my host organism which consists of 3123 contigs. I did all the UMITools preprocessing requested and my package versions are r/4.0.2 samtools/1.10 star/2.7.8a stringtie/2.1.3 plus R packages as follows:

> packageVersion("Biostrings")
[1] ‘2.56.0’
> packageVersion("ShortRead")
[1] ‘1.46.0’
> packageVersion("doParallel")
[1] ‘1.0.16’
> packageVersion("GenomicAlignments")
[1] ‘1.24.0’
> packageVersion("Gviz")
[1] ‘1.32.0’
> packageVersion("GenomicFeatures")
[1] ‘1.40.1’
> packageVersion("Rsubread")
[1] ‘2.2.6’

Please let me know if I can provide any other information, and I look forward to hearing from you!

[PierreBSC]

Hi Zoé,

I might have an idea of where the problem is coming from : in Viral-Track, chromosomes from the host are removed (line 307). The problem is that the name of those chromosomes can change based on the reference genome/species. I would recommend to update the object "Chromosome_to_remove" in line 283 accordingly with the correct chromosome names !

Hope this will work !

Best

Pierre

[14zac2]

Hi Pierre,

Thanks so much for the tip, it worked! It might be worth it to update the code to be able to accommodate multiple hosts. For anyone else referencing this issue, I fixed the script (for my situation) with a few lines of bash code and my host genome fasta file:

# Create list of chromosomes from my fasta file (host.fa)
grep ">" host.fa > chroms.txt
sed -i 's/>//g' chroms.txt
awk '{ print "\""$0"\""}' chroms.txt > chromQuote.txt
paste -s -d, chromQuote.txt > chromQuoteComma.txt
awk '{ print "Chromosome_to_remove = c("$0")"}' chromQuoteComma.txt > formattedChromList.txt
# The following assumes the Viral-Track code is located in ~
# Inserts the file I just created into the appropriate section of the code
sed -e '/KI270394.1/r formattedChromList.txt' ~/Viral-Track/Viral_Track_scanning.R > ~/Viral-Track/Viral_Track_scanning_2.R
# Now run as normal but call on Viral_Track_scanning_2.R

Many thanks again! Zoe

[GhobrialMoheb]

@PierreBSC , @14zac2 Hi Zoe, thanks alot for the update, I have tried what you mentioned and I am getting another error:

Aug 04 12:43:51 ..... started STAR run Aug 04 12:43:51 ..... loading genome Aug 04 12:45:00 ..... started 1st pass mapping Aug 04 12:45:24 ..... finished 1st pass mapping Aug 04 12:45:25 ..... inserting junctions into the genome indices Aug 04 12:47:00 ..... started mapping Aug 04 12:47:26 ..... finished mapping Aug 04 12:47:31 ..... started sorting BAM Aug 04 12:47:39 ..... finished successfully Mapping ofhgmm_100_R2_extracted.fastq done ! All fastq files have been mapped successfully Starting the BAM file analysis Indexing of the bam file for hgmm_100_R2_extracted is done Computing stat file for the bam file for hgmm_100_R2_extracted is done Checking the mapping quality of each virus... Export of the viral SAM file done for hgmm_100_R2_extracted Error in { : task 1 failed - "different row counts implied by arguments" Calls: %dopar% -> Execution halted

May you please help me with the issue?