cummeRbund error in loading cuffdiff gene file
Hello,
I am trying to load my cuffdiff genes tracking file into cummeRbund and I get an error:
> refCuffdiff="/shared/AdrianP/home112013/glomus/RNA-Meiosis/JGI_annot/diff-cross-solo"
> gtfFilePath="/shared/AdrianP/home112013/glomus/RNA-Meiosis/JGI_annot/merged/merged.gtf"
> genomePath="/shared/AdrianP/home112013/glomus/RNA-Meiosis/JGI_annot/Gloin1_AssemblyScaffolds_Repeatmasked.fa"
> cuff <- readCufflinks(dir=refCuffdiff,rebuild=T,gtfFile=gtfFilePath,genome=genomePath)
Creating database /shared/AdrianP/home112013/glomus/RNA-Meiosis/JGI_annot/diff-cross-solo/cuffData.db
Reading Run Info File /shared/AdrianP/home112013/glomus/RNA-Meiosis/JGI_annot/diff-cross-solo/run.info
Writing runInfo Table
Reading Read Group Info /shared/AdrianP/home112013/glomus/RNA-Meiosis/JGI_annot/diff-cross-solo/read_groups.info
Writing replicates Table
Reading GTF file
Writing GTF features to 'features' table...
Reading /shared/AdrianP/home112013/glomus/RNA-Meiosis/JGI_annot/diff-cross-solo/genes.fpkm_tracking
Error in scan(file, what, nmax, sep, dec, quote, skip, nlines, na.strings, :
line 1 did not have 17 elements
>
I did not edit the genes.fpkm_tracking, here is how it looks after head:
tracking_id class_code nearest_ref_id gene_id gene_short_name tss_id locus length coverage Crossing_FPKM Crossing_conf_lo Crossing_conf_hi Crossing_status Solo_FPKM Solo_conf_lo Solo_conf_hi Solo_status
XLOC_000001 - - XLOC_000001 fgenesh1_kg.1_#_2_#_remain_c6340,fgenesh1_kg.1_#_3_#_step3_c2667,fgenesh1_pg.1_#_1,gm1.1_g TSS1,TSS2,TSS3 scaffold_1:622-2570 - - 21.9001 0 51.4671 OK 19.3357 0 51.4078 OK
XLOC_000002 - - XLOC_000002 gm1.12_g TSS4 scaffold_10:10-512 - - 0 0 0 OK 0 0 0 OK
XLOC_000003 - - XLOC_000003 gm1.154_g TSS5 scaffold_100:720-1182 - - 0.0767179 0 0.39657 OK 0 0 0 OK
XLOC_000004 - - XLOC_000004 gm1.153_g TSS6 scaffold_100:21-396 - - 0 0 0 OK 0 0 0 OK
XLOC_000005 - - XLOC_000005 fgenesh1_pg.1000_#_1,gm1.1557_g TSS7,TSS8 scaffold_1000:0-2489 - - 2.43942 0 8.08898 OK 2.15353 0 6.67501 OK
XLOC_000006 - - XLOC_000006 gm1.1558_g TSS9 scaffold_1000:3054-3550 - - 2.65628 0 8.96984 OK 5.06294 0 16.4288 OK
XLOC_000007 - - XLOC_000007 MIX1_1_43,e_gw1.10000.1.1,gm1.14515_g TSS10 scaffold_10000:64-1868 - - 0 0 0 OK 0.0292999 0 0.34366 OK
XLOC_000008 - - XLOC_000008 gm1.14516_g TSS11,TSS12 scaffold_10001:164-1237 - - 0 0 0 OK 0 0 0 OK
XLOC_000009 - - XLOC_000009 CE19_2291,CE23_3268,CE26_2185,CE29_2185,CE31_910,CE32_2410,fgenesh1_kg.10002_#_1_#_remain_c4528,fgenesh1_kg.10002_#_2_#_ACTTGA_L001_R1_(paired)_contig_254,fgenesh1_kg.10002_#_3_#_step3_c2559,fgenesh1_pg.10002_#_1 TSS13,TSS14,TSS15,TSS16,TSS17 scaffold_10002:1191-5743 - - 205.934 0 427.865 OK 137.679 0 294.519 OK
XLOC_000010 - - XLOC_000010 fgenesh1_kg.10002_#_4_#_ACTTGA_L001_R1_(paired)_contig_4434 TSS18,TSS19,TSS20 scaffold_10002:1191-5743 - - 89.4953 0 389.998 OK 125.531 0 422.435 OK
XLOC_000011 - - XLOC_000011 CE37_98,gm1.14519_g TSS21,TSS22,TSS23 scaffold_10003:56-3724 - - 3.44577 0 9.32412 OK 4.41548 0 12.7158 OK
XLOC_000012 - - XLOC_000012 gm1.14520_g TSS24 scaffold_10004:23-239 - - 0.726845 0 1.9669 OK 1.33937 0 3.53072 OK
XLOC_000013 - - XLOC_000013 fgenesh1_pg.10005_#_1,gm1.14521_g TSS25 scaffold_10005:192-1657 - - 0 0 0 OK 0 0 0 OK
XLOC_000014 - - XLOC_000014 gm1.14522_g TSS26 scaffold_10006:251-777 - - 0 0 0 OK 0 0 0 OK
XLOC_000015 - - XLOC_000015 gm1.14523_g TSS27 scaffold_10007:900-1003 - - 0 0 0 OK 0 0 0 OK
XLOC_000016 - - XLOC_000016 gm1.14524_g TSS28 scaffold_10008:25-990 - - 0 0 0 OK 0 0 0 OK
XLOC_000017 - - XLOC_000017 fgenesh1_pg.10009_#_1,gm1.14525_g,gm1.14526_g TSS29,TSS30,TSS31 scaffold_10009:1648-3030 - - 0 0 0 OK 0 0 0 OK
XLOC_000018 - - XLOC_000018 CE52_327,fgenesh1_kg.1001_#_1_#_ACTTGA_L001_R1_(paired)_contig_2142,fgenesh1_kg.1001_#_2_#_remain_c11121 TSS32,TSS33,TSS34,TSS35 scaffold_1001:464-2744 - - 50.3672 0 150.486 OK 23.201 0 63.7491 OK
XLOC_000019 - - XLOC_000019 CE56_391,fgenesh1_kg.10010_#_1_#_step3_c842,gm1.14527_g TSS36,TSS37 scaffold_10010:1116-2040 - - 752.921 0 2536.24 OK 1132.62 0 5135 OK
Does anyone have any idea what went wrong?
Adrian
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Ram
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Adrian Pelin
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Alright, this is resolved now!
A quick email from Loyal A. Goff, a cummeRbund dev. hinted at the root of the problem:
Hi Adrian,
Without being able to reproduce the error first hand, my best guess at this point is the '#' characters in your gtf file. I think others have had issues in the past with '#' because R considers everything after this to be a comment. Is there any way that these characters can be removed from the reference gtf and cuffdiff could be rerun?
LoyKColumn 5 has these issues, and so does the gtf file. A quick sed fixed the issue.
Adrian
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can you show your code that you used to read in the cuffdiff output?
I have modified my post to show that.
It does not seem that there's anything wrong with genes.fpkm_tracking... maybe the scan() function is not opening the right file?
You tried making sym-links to a directory (or putting all input files into 1 directory) and then running readCufflinks() in that directory?
I tried the same commands on a different experiment and I was in the same directory when I invoked R and it worked loading the genes tracking file.
Might it be that scan is parsing the file in a wrong way?
Are you sure your genes.fpkm_tracking is tab-delimited? I think it should be tab-delimited for the program to correctly read the file.
The default output is tab delimited, and what I posted is also tab delimited. Maybe it parses some columns incorrectly? THe
gene_short_name column has many commas.