[Samtools-help] Picard CollectRnaSeqMetrics

[George G. <geo...@du...>]

I am attempting to run Picard’s CollectRnaSeqMetrics from the command line with the following command (the java versions is 1.7.0_75):
java -jar programs/picard-tools-1.134/picard.jar CollectRnaSeqMetrics REF_FLAT=genes2.ref CHART_OUTPUT=tmp".pdf" STRAND=NONE INPUT=new.sam OUTPUT=new.metrics.txt
The issue I am encountering is that this will result in bases being counted as “Ribosomal” as well as reporting strand bias info; my understanding was CollectRnaSeqMetrics would not count these values under my stated options.

I have checked the format of my refflat file and my .sam file and everything appears fine (no errors noted through ValidateSamFile). I have provided a few excerpts of my files below. Please help in anyway possible.

George

REFFLAT file:
AT1G01010.1	AT1G01010.1	Chr1	+	3630	5899	3759	5630	6	3630,3995,4485,4705,5173,5438,	3913,4276,4605,5095,5326,5899,
AT1G01020.1	AT1G01020.1	Chr1	-	5927	8737	6914	8666	10	5927,6436,7156,7383,7563,7761,7941,8235,8416,8570,	6263,7069,7232,7450,7649,7835,7987,8325,8464,8737,
AT1G01020.2	AT1G01020.2	Chr1	-	6789	8737	7314	8666	8	6789,7156,7563,7761,7941,8235,8416,8570,	7069,7450,7649,7835,7987,8325,8464,8737,
AT1G01030.1	AT1G01030.1	Chr1	-	11648	13714	11863	12940	2	11648,13334,	13173,13714,
AT1G01040.1	AT1G01040.1	Chr1	+	23145	31227	23518	31079	20	23145,24541,24751,25040,25523,25824,26080,26291,26542,26861,27098,27371,27617,27802,28707,28889,29159,30146,30409,30901,	24451,24655,24962,25435,25743,25997,26203,26452,26776,27012,27281,27533,27713,28431,28805,29080,30065,30311,30816,31227,

SAM file:
@HD	VN:1.0	SO:coordinate
@SQ	SN:Chr1	LN:30427671
@SQ	SN:Chr2	LN:19698289
@SQ	SN:Chr3	LN:23459830
@SQ	SN:Chr4	LN:18585056
@SQ	SN:Chr5	LN:26975502
@RG	ID:L1	PL:illumina	LB:library	SM:sample
@PG	ID:TopHat	VN:1.3.1	CL:/opt/apps/sdg/bin/tophat --no-novel-juncs -G ../../genes_new.name.gtf.txt --output-dir ./top ../../index/a_thaliana ./bt2/MM2_25_nocont.fq
SEQCORE-1795804:191:C6HVDANXX:8:1101:8707:62378	256	Chr1	4293	0	27M	*	0	0	ATATATATATATATATATTTGAGGATA	BBBBBFFFFFFFFFFFFFFFFFFBBFF	NM:i:2	NH:i:14	CC:Z:=	CP:i:10911936	HI:i:0	RG:Z:L1
SEQCORE-1795804:191:C6HVDANXX:7:1201:13268:85214	16	Chr1	8288	255	29M	*	0	0	ACCTTTGGTCTGTGAAGGATTAAATCGAT	FFFFFFFFF<FFFBFFFBFFBFFBBBBBB	NM:i:0	NH:i:1	RG:Z:L1
metrics.txt:
# picard.analysis.CollectRnaSeqMetrics REF_FLAT=genes2.ref STRAND_SPECIFICITY=NONE CHART_OUTPUT=tmp.pdf INPUT=new.sam OUTPUT=new.metrics.txt REFERENCE_SEQUENCE=index/a_thaliana.fasta    MINIMUM_LENGTH=500 RRNA_FRAGMENT_PERCENTAGE=0.8 METRIC_ACCUMULATION_LEVEL=[ALL_READS] ASSUME_SORTED=true STOP_AFTER=0 VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false
## htsjdk.samtools.metrics.StringHeader
# Started on: Tue Jun 23 11:39:48 EDT 2015

## METRICS CLASS	picard.analysis.RnaSeqMetrics
PF_BASES	PF_ALIGNED_BASES	RIBOSOMAL_BASES	CODING_BASES	UTR_BASES	INTRONIC_BASES	INTERGENIC_BASES	IGNORED_READS	CORRECT_STRAND_READS	INCORRECT_STRAND_READS	PCT_RIBOSOMAL_BASES	PCT_CODING_BASES	PCT_UTR_BASES	PCT_INTRONIC_BASES	PCT_INTERGENIC_BASES	PCT_MRNA_BASES	PCT_USABLE_BASES	PCT_CORRECT_STRAND_READS	MEDIAN_CV_COVERAGE	MEDIAN_5PRIME_BIAS	MEDIAN_3PRIME_BIAS	MEDIAN_5PRIME_TO_3PRIME_BIAS	SAMPLE	LIBRARY	READ_GROUP
22349250	22349250		16608264	4455239	266041	1019706	0	0	0		0.743124	0.199346	0.011904	0.045626	0.94247	0.94247	0	3.008882	0.070426	0	0