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

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