I'm not sure why one set of bam files in my dataset is producing the below error. To be safe, I re-trimmed and re-aligned my fastqs and got the same result. Is this something I can suppress somehow? This can't possibly mean length of the bams because all of my bams have a different number of lines and only one set is producing an error.

Error in GAlignmentPairs(ga[first], ga[last], isProperPair = isProperPair, : 'first' and 'last' must have the same length

-------Failing Pair------- BAM
2 @SQ SN:chr1 LN:248956422 @SQ SN:chr2 LN:242193529 @SQ SN:chr3 LN:198295559 @SQ SN:chr4 LN:190214555 @SQ SN:chr5 LN:181538259 @SQ SN:chr6 LN:170805979 @SQ SN:chr7 LN:159345973 @SQ SN:chr8 LN:145138636 @SQ SN:chr9 LN:138394717 @SQ SN:chr10 LN:133797422

Length: 231463

BAM
2 @SQ SN:chr1 LN:248956422 @SQ SN:chr2 LN:242193529 @SQ SN:chr3 LN:198295559 @SQ SN:chr4 LN:190214555 @SQ SN:chr5 LN:181538259 @SQ SN:chr6 LN:170805979 @SQ SN:chr7 LN:159345973 @SQ SN:chr8 LN:145138636 @SQ SN:chr9 LN:138394717 @SQ SN:chr10 LN:133797422

Length: 285842

-------Working Pair------- BAM
2 @SQ SN:chr1 LN:248956422 @SQ SN:chr2 LN:242193529 @SQ SN:chr3 LN:198295559 @SQ SN:chr4 LN:190214555 @SQ SN:chr5 LN:181538259 @SQ SN:chr6 LN:170805979 @SQ SN:chr7 LN:159345973 @SQ SN:chr8 LN:145138636 @SQ SN:chr9 LN:138394717 @SQ SN:chr10 LN:133797422

Length: 206059

BAM
2 @SQ SN:chr1 LN:248956422 @SQ SN:chr2 LN:242193529 @SQ SN:chr3 LN:198295559 @SQ SN:chr4 LN:190214555 @SQ SN:chr5 LN:181538259 @SQ SN:chr6 LN:170805979 @SQ SN:chr7 LN:159345973 @SQ SN:chr8 LN:145138636 @SQ SN:chr9 LN:138394717 @SQ SN:chr10 LN:133797422

Length: 76069

-Tomás

Tom,

Could you please send me the set of bam files and the corresponding UMI files to reproduce the error ? Thanks!

Best regards,

Julie

Tomás, I am able to run the analysis with your data without any issue. Here is my sessionInfo and the test script. I would be happy to test your script if you could share it with me. Best regards, Julie

library("BSgenome.Hsapiens.UCSC.hg38") library("org.Hs.eg.db") library(("TxDb.Hsapiens.UCSC.hg38.knownGene")

         umiFile <- c("UMIs.txt", "UMIs.txt")
         alignFile <- c("WT_SpyCas9-STS4.bam",  "neg_WT_SpyCas9-STS4.bam")
         gRNA.file <- "2guide.fa"

         guideSeqRes <- GUIDEseqAnalysis(
             alignment.inputfile = alignFile,
             umi.inputfile = umiFile, gRNA.file = gRNA.file,
             orderOfftargetsBy = "peak_score",
             descending = TRUE,
             keepTopOfftargetsBy = "predicted_cleavage_score",
             scoring.method = "CFDscore", orgAnn = org.Hs.egSYMBOL, 
    txdb = TxDb.Hsapiens.UCSC.hg38.knownGene,
             BSgenomeName = Hsapiens, min.reads = 1, n.cores.max = 1)
         guideSeqRes$offTargets
         names(guideSeqRes)

sessionInfo() R version 3.6.1 (2019-07-05) Platform: x86_64-apple-darwin15.6.0 (64-bit) Running under: macOS Sierra 10.12.6

Matrix products: default BLAS: /Library/Frameworks/R.framework/Versions/3.6/Resources/lib/libRblas.0.dylib LAPACK: /Library/Frameworks/R.framework/Versions/3.6/Resources/lib/libRlapack.dylib

locale: [1] enUS.UTF-8/enUS.UTF-8/enUS.UTF-8/C/enUS.UTF-8/en_US.UTF-8

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

other attached packages: [1] TxDb.Hsapiens.UCSC.hg38.knownGene3.4.6 [2] GenomicFeatures1.36.4
[3] BiocManager1.30.4
[4] org.Hs.eg.db3.8.2
[5] AnnotationDbi1.46.0
[6] Biobase2.44.0
[7] BSgenome.Hsapiens.UCSC.hg381.4.1
[8] BSgenome.Mmusculus.UCSC.mm101.4.0
[9] BSgenome.Hsapiens.UCSC.hg191.4.0
[10] BSgenome1.52.0
[11] rtracklayer1.44.2
[12] Biostrings2.52.0
[13] XVector0.24.0
[14] GUIDEseq1.14.0
[15] GenomicRanges1.36.0
[16] GenomeInfoDb1.20.0
[17] IRanges2.18.1
[18] S4Vectors0.22.0
[19] BiocGenerics_0.30.0

loaded via a namespace (and not attached): [1] httr1.4.1 idr1.2
[3] regioneR1.16.2 bit640.9-7
[5] splines3.6.1 assertthat0.2.1
[7] RBGL1.60.0 blob1.2.0
[9] GenomeInfoDbData1.2.1 Rsamtools2.0.0
[11] progress1.2.2 pillar1.4.2
[13] RSQLite2.1.2 backports1.1.4
[15] lattice0.20-38 limma3.40.6
[17] digest0.6.20 Matrix1.2-17
[19] XML3.98-1.20 pkgconfig2.0.2
[21] biomaRt2.40.3 zlibbioc1.30.0
[23] GO.db3.8.2 ChIPpeakAnno3.18.2
[25] VennDiagram1.6.20 BiocParallel1.18.0
[27] tibble2.1.3 AnnotationFilter1.8.0
[29] SummarizedExperiment1.14.1 lazyeval0.2.2
[31] survival2.44-1.1 magrittr1.5
[33] crayon1.3.4 memoise1.1.0
[35] MASS7.3-51.4 graph1.62.0
[37] tools3.6.1 hash2.2.6.1
[39] data.table1.12.2 prettyunits1.0.2
[41] hms0.5.0 formatR1.7
[43] matrixStats0.54.0 stringr1.4.0
[45] DelayedArray0.10.0 lambda.r1.2.3
[47] ensembldb2.8.0 ade41.7-13
[49] compiler3.6.1 rlang0.4.0
[51] futile.logger1.4.3 grid3.6.1
[53] RCurl1.95-4.12 CRISPRseek1.24.0
[55] tcltk3.6.1 bitops1.0-6
[57] multtest2.40.0 DBI1.0.0
[59] curl4.0 R62.4.0
[61] GenomicAlignments1.20.1 seqinr3.4-5
[63] bit1.1-14 zeallot0.1.0
[65] ProtGenerics1.16.0 futile.options1.0.1
[67] stringi1.4.3 Rcpp1.0.2
[69] vctrs_0.2.0