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20 hours ago •
al2na
al2na
Issues with processBismarkAln()
miyakokodama commented 3 minutes ago Hi,
I am new to methylKit, I am currently trying to read my bam files with processBismarkAln however I get an error message I don't understand.
setwd("/home/projects/5_bismark")
file.list=list("BM_174/BM_174.pair1.truncated.gz_bismark_bt2_pe_sorted.bam",
"BM_427/BM_427.pair1.truncated.gz_bismark_bt2_pe_sorted.bam",
"BM_437/BM_437.pair1.truncated.gz_bismark_bt2_pe_sorted.bam",
"BM_455/BM_455.pair1.truncated.gz_bismark_bt2_pe_sorted.bam",
"E_220/E_220.pair1.truncated.gz_bismark_bt2_pe_sorted.bam",
"E_256/E_256.pair1.truncated.gz_bismark_bt2_pe_sorted.bam",
"E_265/E_265.pair1.truncated.gz_bismark_bt2_pe_sorted.bam",
"E_274/E_274.pair1.truncated.gz_bismark_bt2_pe_sorted.bam")
CpG.bismark.data <- processBismarkAln(location=file.list, sample.id = list("BM1","BM2","BM3","BM4","E1","E2","E3","E4"), treatment =c(0,0,0,0,1,1,1,1), assembly = "GCF_000233375.1_ICSASG_v2_genomic", save.folder=getwd(), save.context = c("CpG"), read.context="CpG", nolap=FALSE, mincov=10, minqual=20)
Once these are typed in, I get a following message.
/home/projects/5_bismark/BM_174/BM_174.pair1.truncated.gz_bismark_bt2_pe_sorted.bam
using htslib.
Error in methCall(read1 = location, type = "bam", nolap = nolap, minqual = minqual, :
basic_string::_M_construct null not valid
Any hints/advice you could provide would be greatly appreciated.
Miyako
Hi Miyako,
Could you please send your session info?
Also please verify that all your bamfiles are fine, maybe with samtools quickcheck .
Best, Alex
Please also provide a small reproducible example, because otherwise I cannot test for your error.
Hi Alex, thanks for the reply! I will get back to you as soon as I can.
Hi, Here is the sessioninfo():
> sessionInfo()
R version 3.6.1 (2019-07-05)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: CentOS Linux 7 (Core)
Matrix products: default
BLAS/LAPACK: /services/tools/intel/perflibs/2019_update5/compilers_and_libraries_2019.5.281/linux/mkl/lib/intel64_lin/libmkl_gf_lp64.so
locale:
[1] LC_CTYPE=en_DK.UTF-8 LC_NUMERIC=C
[3] LC_TIME=en_DK.UTF-8 LC_COLLATE=en_DK.UTF-8
[5] LC_MONETARY=en_DK.UTF-8 LC_MESSAGES=en_DK.UTF-8
[7] LC_PAPER=en_DK.UTF-8 LC_NAME=C
[9] LC_ADDRESS=C LC_TELEPHONE=C
[11] LC_MEASUREMENT=en_DK.UTF-8 LC_IDENTIFICATION=C
attached base packages:
[1] parallel stats4 stats graphics grDevices utils datasets
[8] methods base
other attached packages:
[1] methylKit_1.12.0 GenomicRanges_1.38.0 GenomeInfoDb_1.22.0
[4] IRanges_2.20.2 S4Vectors_0.24.3 BiocGenerics_0.32.0
[7] RCurl_1.95-4.12 bitops_1.0-6 devtools_2.2.2
[10] usethis_1.5.1 sessioninfo_1.1.1 memoise_1.1.0
[13] BiocManager_1.30.10 RLinuxModules_0.2
loaded via a namespace (and not attached):
[1] Biobase_2.46.0 pkgload_1.0.2
[3] splines_3.6.1 R.utils_2.9.2
[5] gtools_3.8.1 assertthat_0.2.1
[7] GenomeInfoDbData_1.2.2 Rsamtools_2.2.3
[9] remotes_2.1.1 numDeriv_2016.8-1.1
[11] pillar_1.4.4 backports_1.1.6
[13] lattice_0.20-40 glue_1.4.0
[15] limma_3.42.2 bbmle_1.0.23.1
[17] digest_0.6.25 XVector_0.26.0
[19] qvalue_2.18.0 colorspace_1.4-1
[21] Matrix_1.2-18 R.oo_1.23.0
[23] plyr_1.8.6 XML_3.98-1.20
[25] pkgconfig_2.0.3 emdbook_1.3.12
[27] zlibbioc_1.32.0 purrr_0.3.4
[29] mvtnorm_1.1-0 scales_1.1.0
[31] processx_3.4.2 BiocParallel_1.20.1
[33] tibble_3.0.1 ggplot2_3.3.0
[35] ellipsis_0.3.0 SummarizedExperiment_1.16.1
[37] withr_2.2.0 cli_2.0.2
[39] mclust_5.4.5 magrittr_1.5
[41] crayon_1.3.4 ps_1.3.2
[43] R.methodsS3_1.8.0 fs_1.3.2
[45] fansi_0.4.1 MASS_7.3-51.5
[47] pkgbuild_1.0.7 tools_3.6.1
[49] data.table_1.12.8 prettyunits_1.1.1
[51] matrixStats_0.56.0 lifecycle_0.2.0
[53] stringr_1.4.0 munsell_0.5.0
[55] DelayedArray_0.12.2 callr_3.4.3
[57] Biostrings_2.54.0 compiler_3.6.1
[59] fastseg_1.32.0 rlang_0.4.6
[61] grid_3.6.1 testthat_2.3.2
[63] gtable_0.3.0 reshape2_1.4.3
[65] R6_2.4.1 GenomicAlignments_1.22.1
[67] rtracklayer_1.46.0 dplyr_0.8.5
[69] bdsmatrix_1.3-4 rprojroot_1.3-2
[71] desc_1.2.0 stringi_1.4.6
[73] Rcpp_1.0.4.6 vctrs_0.2.4
[75] tidyselect_1.0.0 coda_0.19-3
I also did samtools quickcheck but no errors were detected. However, when I used picard ValidateSamFile, I got the following error:
WARNING 2020-05-13 13:20:33 ValidateSamFile NM validation cannot be performed without the reference. All other validations will still occur.
ERROR: Read groups is empty
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:1090:1000_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:1090:1000_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:24306:1031_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:24306:1031_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:17526:1094_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:17526:1094_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:9182:1110_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:9182:1110_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:26738:1110_1:N:0:TGTCTAGA+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:26738:1110_1:N:0:TGTCTAGA+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:22064:1125_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:22064:1125_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:3251:1141_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:3251:1141_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:3369:1157_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:3369:1157_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:28492:1172_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:28492:1172_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:11993:1188_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:11993:1188_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:6090:1204_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:6090:1204_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:26756:1204_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:26756:1204_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:4065:1235_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:4065:1235_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:8901:1282_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:8901:1282_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:27525:1282_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:27525:1282_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:27615:1313_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:27615:1313_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:21603:1329_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:21603:1329_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:15519:1344_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:15519:1344_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:7392:1360_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:7392:1360_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:24985:1360_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:24985:1360_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:24722:1376_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:24722:1376_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:14353:1391_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:14353:1391_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:2031:1407_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:2031:1407_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:22263:1438_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:22263:1438_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:13774:1454_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:13774:1454_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:10583:1470_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:10583:1470_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:12373:1501_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:12373:1501_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:19036:1517_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:19036:1517_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:2736:1532_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:2736:1532_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:11677:1548_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:11677:1548_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:31367:1548_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:31367:1548_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:23610:1579_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:23610:1579_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:1542:1626_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:1542:1626_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:13892:1626_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:13892:1626_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:31286:1626_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:31286:1626_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:6451:1705_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:6451:1705_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:14742:1720_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:14742:1720_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:25283:1752_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:25283:1752_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:21296:1767_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:21296:1767_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:3766:1783_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:3766:1783_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:13883:1799_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:13883:1799_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:22354:1814_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:22354:1814_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:12599:1830_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:12599:1830_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:14543:1846_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:14543:1846_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:15980:1861_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:15980:1861_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:27028:1861_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:27028:1861_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:1316:1892_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:1316:1892_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:15447:1908_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:15447:1908_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:16206:1939_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:16206:1939_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
WARNING: Read name A01045:96:HWNN7DSXX:4:1101:31286:1939_1:N:0:TGTCTAGC+GCTACTCA, A record is missing a read group
Maximum output of [100] errors reached.
I would also like to add that processBismarkAln() produces an output ("CpG.txt" and "CpG_conversionStats.txt" files) for the first sample, and then quits with the error I mentioned in the first post. The "CpG.txt" seems to be truncated.
If any of these errors don't ring a bell, please let me know - I will try to subset my .bam files and provide a small reproducible example.
Many thanks for your help in advance!
Miyako
Hi @miyakokodama ,
please provide me a subset of bam files that causes the error, because this does not seem to be an obvious error. I cannot say wether this is a bug in the code or wether something is wrong with your samples unless I have the small reproducible example.
Best, Alex
Hi, dear @alexg9010 @miyakokodama , Now I also suffer this issue with processBismarkAln(), so could you please share the solution with me again?
