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Old 01-30-2012, 08:11 AM   #1
ckrna
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Default Picard Tools "loaded 0 genes"

I am testing the CollectRnaSeqMetrics script in Picard Tools for the first time and received the following error message:


INFO Date Time CollectRnaSeqMetrics Loaded 0 genes.

Then the output begins to show “Processed 1,000,000 records” and so on. The file created at the end of the process includes numbers for PF_Bases, PF_Aligned_Bases, and Intergenic_bases but all other fields (Ribosomal_bases, coding_bases, pct_UTR_bases, etc) contain 0’s.

What am I doing wrong? All help is appreciated, thanks.
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Old 01-30-2012, 08:52 PM   #2
Jon_Keats
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Likely the reflat file and/or ribosome reference file is not formatted correctly. Which genome are you aligning against?

reflat example
Code:
#geneName	name	chrom	strand	txStart	txEnd	cdsStart	cdsEnd	exonCount	exonStarts	exonEnds
WASH7P	NR_024540	chr1	-	14361	29370	29370	29370	11	14361,14969,15795,16606,16857,17232,17605,17914,18267,24737,29320,	14829,15038,15947,16765,17055,17368,17742,18061,18366,24891,29370,
FAM138A	NR_026818	chr1	-	34610	36081	36081	36081	3	34610,35276,35720,	35174,35481,36081,
FAM138F	NR_026820	chr1	-	34610	36081	36081	36081	3	34610,35276,35720,	35174,35481,36081,
OR4F5	NM_001005484	chr1	+	69090	70008	69090	70008	1	69090,	70008,
LOC729737	NR_039983	chr1	-	136697	140566	140566	140566	5	136697,136805,136952,139789,140074,	136756,136854,139696,139847,140566,
LOC100132287	NR_028322	chr1	+	323891	328581	328581	328581	3	323891,324287,324438,	324060,324345,328581,
LOC100133331	NR_028327	chr1	+	323891	328581	328581	328581	4	323891,324287,324438,327035,	324060,324345,326938,328581,
LOC100132062	NR_028325	chr1	+	323891	328581	328581	328581	3	323891,324287,324438,	324060,324345,328581,
OR4F3	NM_001005224	chr1	+	367658	368597	367658	368597	1	367658,	368597,
OR4F16	NM_001005277	chr1	+	367658	368597	367658	368597	1	367658,	368597,
OR4F29	NM_001005221	chr1	+	367658	368597	367658	368597	1	367658,	368597,
OR4F16	NM_001005277	chr1	-	621095	622034	621095	622034	1	621095,	622034,
ribosome location file
Code:
@SQ	SN:chr1	LN:249250621
@SQ	SN:chr10	LN:135534747
@SQ	SN:chr11	LN:135006516
@SQ	SN:chr12	LN:133851895
@SQ	SN:chr13	LN:115169878
@SQ	SN:chr14	LN:107349540
@SQ	SN:chr15	LN:102531392
@SQ	SN:chr16	LN:90354753
@SQ	SN:chr17	LN:81195210
@SQ	SN:chr18	LN:78077248
@SQ	SN:chr19	LN:59128983
@SQ	SN:chr2	LN:243199373
@SQ	SN:chr20	LN:63025520
@SQ	SN:chr21	LN:48129895
@SQ	SN:chr22	LN:51304566
@SQ	SN:chr3	LN:198022430
@SQ	SN:chr4	LN:191154276
@SQ	SN:chr5	LN:180915260
@SQ	SN:chr6	LN:171115067
@SQ	SN:chr7	LN:159138663
@SQ	SN:chr8	LN:146364022
@SQ	SN:chr9	LN:141213431
@SQ	SN:chrM	LN:16571
@SQ	SN:chrX	LN:155270560
@SQ	SN:chrY	LN:59373566
chr18	9844714	9844843	-	ENSG00000252680
chr18	9923917	9924018	+	ENSG00000223138
chr18	19508946	19509055	-	ENSG00000222520
chr18	21191674	21191827	+	ENSG00000240442
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Old 02-08-2012, 12:08 PM   #3
ckrna
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Default Thanks!

Hi Jon,

You are correct - formatting is the problem. I am aligning to hg19. The bam file does not include the "chr" in "chr1". Any suggestions on how to change the formatting of an existing file or recreate the file with the proper formatting?

Thanks!
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Old 02-08-2012, 12:26 PM   #4
Jon_Keats
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If the bam file does not contain chr under the chromosome column then you didn't align to hg19 downloaded from UCSC. Sometimes people get the reference file from say ensembl but then rename it hg19 because they don't like the long description used. This obviously is bad practice as there are subtle differences in the two version chr1 vs 1 and chrM vs MT. If you want do a "samtools view -H YOURBAMFILE.bam" post it or PM me and if I have the matching genome I'll send you the required files that should work.
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