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Question: Splicemap (in QuasR) failed while aligning 25mers
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gravatar for Gabriella
19 months ago by
Gabriella0
Gabriella0 wrote:

Hello,

I am using QuasR to do spliced alignment of paired end reads to the human genome. This is done through SpliceMap. I have received an error message, which I have been unable to find in any forums. Is it possible for anyone to assist me?

Here is the command that I used:

alignmentproject <- qAlign(sampleFile,genome=genomeName, paired="fr", splicedAlignment=TRUE, cacheDir=tempdir, clObj=cl)

My read length is 76, if this is relevant. It occurred to me that the problem could be short reads, since Splicemap does not work for short reads, but I do not think that this is the issue.

Thank you so much for your assistance! This is my first time going through the RNA-seq analysis pipeline, and this forum has been invaluable.

Cheers,

Gabriella

 

The log message is as follows:

[1] "Decompressing file on Theory9 : /home/Lab/Desktop/Gabriella/RNA_Seq/Epidermis/Basal_Ker_S2_R1_001.fastq.gz" [1] "Decompressing file on Theory9 : /home/Lab/Desktop/Gabriella/RNA_Seq/Epidermis/Basal_Ker_S2_R2_001.fastq.gz" [1] "Writing BSgenome to disk on Theory9 : /home/Lab/Desktop/Gabriella/RNA_Seq/Epidermis/TempDir/RtmpKW69pw/file28337981ede.fa" [1] "Executing Splicemap on Theory9 using 6 cores. Parameters:" [1] "-max_intron 400000 -min_intron 20000 -max_multi_hit 10 -selectSingleHit TRUE -seed_mismatch 1 -read_mismatch 2 -try_hard yes -genome_dir /home/Lab/Desktop/Gabriella/RNA_Seq/Epidermis/TempDir/RtmpKW69pw/file28337981ede.fa -reads_list1 /home/Lab/Desktop/Gabriella/RNA_Seq/Epidermis/TempDir/RtmpKW69pw/Basal_Ker_S2_R1_001.fastq.gz2833a4169e3.fastq -reads_list2 /home/Lab/Desktop/Gabriella/RNA_Seq/Epidermis/TempDir/RtmpKW69pw/Basal_Ker_S2_R2_001.fastq.gz283311e34d93.fastq -read_format FASTQ -quality_format phred-33 -temp_path /home/Lab/Desktop/Gabriella/RNA_Seq/Epidermis/TempDir/RtmpKW69pw -num_chromosome_together 6 -num_threads 6 -bowtie_base_dir /home/Lab/R/x86_64-redhat-linux-gnu-library/3.2/BSgenome.Hsapiens.UCSC.hg19.Rbowtie/alignmentIndex/bowtieIndex -outfile /home/Lab/Desktop/Gabriella/RNA_Seq/Epidermis/TempDir/RtmpKW69pw/Basal_Ker_S2_R1_001.fastq.gz2833a4169e3.fastq2833355d17a6.sam" [1] "Error on Theory9 processing sample /home/Lab/Desktop/Gabriella/RNA_Seq/Epidermis/TempDir/RtmpKW69pw/Basal_Ker_S2_R1_001.fastq.gz2833a4169e3.fastq : failed while aligning 25mers"

Here is my session info:

R version 3.2.3 (2015-12-10) Platform: x86_64-redhat-linux-gnu (64-bit) Running under: CentOS release 6.7 (Final)

locale: [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
[3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
[5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
[7] LC_PAPER=en_US.UTF-8 LC_NAME=C
[9] LC_ADDRESS=C LC_TELEPHONE=C
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C

attached base packages: [1] stats4 parallel stats graphics grDevices utils datasets [8] methods base

other attached packages: [1] BSgenome.Hsapiens.UCSC.hg19_1.4.0 BSgenome_1.38.0
[3] rtracklayer_1.30.2 Biostrings_2.38.4
[5] XVector_0.10.0 QuasR_1.10.1
[7] Rbowtie_1.10.0 GenomicRanges_1.22.4
[9] GenomeInfoDb_1.6.3 IRanges_2.4.8
[11] S4Vectors_0.8.11 BiocGenerics_0.16.1
[13] BiocInstaller_1.20.1

loaded via a namespace (and not attached): [1] AnnotationDbi_1.32.3 GenomicAlignments_1.6.3
[3] zlibbioc_1.16.0 BiocParallel_1.4.3
[5] lattice_0.20-33 hwriter_1.3.2
[7] tools_3.2.3 grid_3.2.3
[9] SummarizedExperiment_1.0.2 Biobase_2.30.0
[11] DBI_0.3.1 latticeExtra_0.6-28
[13] lambda.r_1.1.7 futile.logger_1.4.1
[15] GenomicFiles_1.6.2 RColorBrewer_1.1-2
[17] futile.options_1.0.0 bitops_1.0-6
[19] biomaRt_2.26.1 RCurl_1.95-4.8
[21] RSQLite_1.0.0 GenomicFeatures_1.22.13
[23] Rsamtools_1.22.0 ShortRead_1.28.0
[25] XML_3.98-1.4
ADD COMMENTlink modified 19 months ago • written 19 months ago by Gabriella0

Hi Gabriella

 

Just a comment so far:

Did you run "preprocessReads" on you fastq files?

Also, have you tried qAlign with "splicedAlignment=TRUE" but not paired, i.e. the two fastq files (Basal_Ker_S2_R1_001.fastq.gz, Basal_Ker_S2_R2_001.fastq.gz) as independent samples

 

Regards, Hans-Rudolf

 

 

 

 

ADD REPLYlink written 19 months ago by Hotz, Hans-Rudolf380
0
gravatar for Gabriella
19 months ago by
Gabriella0
Gabriella0 wrote:

Hello Hans-Rudolf,

Thank you so much for your assistance. I did not run preprocessReads on the fastq files. Would it be recommended to try? I was able to successfully align using paired=NULL, splicedAlignment=TRUE.

Thanks again!

Gabriella

ADD COMMENTlink written 19 months ago by Gabriella0

Hi Gabriella

I was asking about the preprocessReads, since this could have been the source the problem. As you did not use it, we can rule it out.

IMHO, the spliced alignments in QuasR are a 'challange' . Would you mind sharing a reproducible example with me? and I will try to look deeper into it and/or provide you with a solution.

Regards, Hans-Rudolf

 

ADD REPLYlink written 19 months ago by Hotz, Hans-Rudolf380

Hello Hans-Rudolf,

Thanks again for your help. Before constructing a reproducible example, I tried using the same commands to produce a spliced, unpaired alignment for a different file from the same experiment, which should be similar in every way. However, I obtained the same error message as above. This makes me think that perhaps it is not the paired-end condition which is inducing the error. Any thoughts? I will try to create a reproducible example so we can troubleshoot further.

Thanks again,

Gabriella

ADD REPLYlink modified 19 months ago • written 19 months ago by Gabriella0
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