Piccard module(CollectRNASeqMetrics) error

Question

[Biocode_user]

Hello,

I am trying to get the mapping statistics from my bam file and I am mainly interested to look into how many reads map to cds, 5' and 3' UTR's. I a trying to use CollectRNASeqMetrics and I get the following error

I used gtfToGenePred to convert my gtf file to refFlat file. and my refFlat looks like

Ec-00_000010.1  chr_00  -       149     6731    149     6731    10      149,897,1535,2091,2535,3474,4006,4702,6245,6709,        428,1100,1674,2268,3070,3557,4155,4968,6363,6731,
Ec-00_000020.1  chr_00  -       28572   29122   28572   29122   2       28572,28937,    28582,29122,
Ec-00_000030.1  chr_00  +       29412   32214   29412   32214   1       29412,  32214,
Ec-00_000040.1  chr_00  +       34287   34360   34287   34360   1       34287,  34360,
Ec-00_000050.1  chr_00  -       36705   39329   36705   37902   3       36705,37422,39143,      36870,37944,39329,
Ec-00_000060.1  chr_00  +       43007   44099   43007   44099   3       43007,43404,43829,      43046,43455,44099,

and when I try to run

java -jar /software/shared/apps/x86_64/picard-tools/1.56/CollectRnaSeqMetrics.jar \
  REF_FLAT=chr_00.refFlat.txt \
  RIBOSOMAL_INTERVALS=null \
  STRAND_SPECIFICITY= NONE \
  CHART_OUTPUT=b2_cOLLECTrna.pdf \
  METRIC_ACCUMULATION_LEVEL=ALL_READS \
  INPUT=out.prefix.bam \
  OUTPUT=B2_CollectRNAMetrices

I end up getting

Exception in thread "main" net.sf.picard.annotation.AnnotationException: Wrong number of fields in refFlat file chr_00.refFlat.txt at line 1
        at net.sf.picard.annotation.RefFlatReader.load(RefFlatReader.java:80)
        at net.sf.picard.annotation.RefFlatReader.load(RefFlatReader.java:66)
        at net.sf.picard.annotation.GeneAnnotationReader.loadRefFlat(GeneAnnotationReader.java:37)
        at net.sf.picard.analysis.CollectRnaSeqMetrics.setup(CollectRnaSeqMetrics.java:137)
        at net.sf.picard.analysis.SinglePassSamProgram.makeItSo(SinglePassSamProgram.java:101)
        at net.sf.picard.analysis.SinglePassSamProgram.doWork(SinglePassSamProgram.java:54)
        at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:175)
        at net.sf.picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:118)
        at net.sf.picard.analysis.CollectRnaSeqMetrics.main(CollectRnaSeqMetrics.java:101

Could anyone help me figure out what wrong with Refflat file. Is there any problem with my gtf istelf?

Thanks

Answer 1

[cjsifuen]

I know this is late, so you probably figured it out already, but it looks like you're missing a column. You need a gene name and transcript id. I am not sure which one of those the first column that you already have is, but you seem to be missing the other.