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
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