Hi,
I am having a problem with RIBOSOMAL_BASES estimated as 0.
When I open the mapped reads I observe several reads at ribosomal positions.
The same problem is discussed in another forum but no solution is described: https://sourceforge.net/p/samtools/mailman/message/29445029/
I already search for a similar problem in FAQ and GATK forum and also run ValidateSamFile.
The chromosome names are the same in the refFlat file, BAM and interval_list.
Can anyone help me?
Command line:
java -jar picard.jar CollectRnaSeqMetrics I=input--bowtie2-RG.sorted.fixed_mate.bam O=output--bowtie2-RG.sorted.fixed_mate-Second_Strand-Metrics REF_FLAT=LbrM2903--flatfile.txt STRAND=SECOND_READ_TRANSCRIPTION_STRAND RIBOSOMAL_INTERVALS=rRNAs_LbrM2903.interval_list
refFlat file excerpt:
LBRM2903_000009300 LBRM2903_000009300 LBRM2903_SCAF000663 - 102 434 102 434 1 102 434
LBRM2903_000009400 LBRM2903_000009400 LBRM2903_SCAF000674 - 285 725 285 725 1 285 725
LBRM2903_000009500 LBRM2903_000009500 LBRM2903_SCAF000686 - 544 1026 544 1026 1 544 1026
LBRM2903_000009600 LBRM2903_000009600 LBRM2903_SCAF000689 - 158 1174 158 1174 1 158 1174
LBRM2903_000009700 LBRM2903_000009700 LBRM2903_SCAF000701 - 11 208 11 208 1 11 208
LBRM2903_000009800 LBRM2903_000009800 LBRM2903_SCAF000702 - 147 1901 147 1901 1 147 1901
LbrM2903_010005000 LbrM2903_010005000 LbrM2903_01 - 3897 4895 3897 4895 1 3897 4895
LbrM2903_010005100 LbrM2903_010005100 LbrM2903_01 - 5941 7602 5941 7602 1 5941 7602
LbrM2903_010005200 LbrM2903_010005200 LbrM2903_01 - 9153 11144 9153 11144 1 9153 11144
LbrM2903_010005300 LbrM2903_010005300 LbrM2903_01 - 12123 12683 12123 12683 1 12123 12683
LbrM2903_010005400 LbrM2903_010005400 LbrM2903_01 - 14878 16875 14878 16875 1 14878 16875
LbrM2903_010005500 LbrM2903_010005500 LbrM2903_01 - 18129 18845 18129 18845 1 18129 18845
LbrM2903_010005600 LbrM2903_010005600 LbrM2903_01 - 19985 21838 19985 21838 1 19985 21838
LbrM2903_010005700 LbrM2903_010005700 LbrM2903_01 - 22674 23366 22674 23366 1 22674 23366
intervals_list file excerpt:
@SQ SN:LbrM2903_32 LN:1666992
@SQ SN:LbrM2903_33 LN:1581728
@SQ SN:LbrM2903_34 LN:2192442
@SQ SN:LbrM2903_35 LN:2890053
LbrM2903_06 328796 331000 + LbrM2903_06_rRNA.01
LbrM2903_06 331200 331390 + LbrM2903_06_rRNA.07
LbrM2903_06 331921 333701 + LbrM2903_06_rRNA.13
LbrM2903_06 333757 333968 + LbrM2903_06_rRNA.19
LbrM2903_06 334128 335652 + LbrM2903_06_rRNA.25
LbrM2903_06 335704 335864 + LbrM2903_06_rRNA.31
LbrM2903_06 336365 336437 + LbrM2903_06_rRNA.37
LbrM2903_06 336668 336796 + LbrM2903_06_rRNA.43
LbrM2903_09 439672 439790 + LbrM2903_09_5SrRNA.01
LbrM2903_09 456487 456602 - LbrM2903_09_5SrRNA.02
LbrM2903_11 1421 1543 - LbrM2903_11_5SRRNA.01
LbrM2903_11 9103 9225 - LbrM2903_11_5SRRNA.02
BAM - samtools view -H:
@SQ SN:LBRM2903_SCAF000705 LN:1284
@SQ SN:LBRM2903_SCAF000706 LN:398
@SQ SN:LBRM2903_SCAF000707 LN:353
@SQ SN:LBRM2903_SCAF000708 LN:560
@SQ SN:LBRM2903_SCAF000709 LN:905
@SQ SN:LbrM2903_01 LN:286661
@SQ SN:LbrM2903_02 LN:352826
@SQ SN:LbrM2903_03 LN:408717
@SQ SN:LbrM2903_04 LN:502955
@SQ SN:LbrM2903_05 LN:505114
@SQ SN:LbrM2903_06 LN:590413
@SQ SN:LbrM2903_07 LN:639434
@SQ SN:LbrM2903_08 LN:537514
@SQ SN:LbrM2903_09 LN:620965
@SQ SN:LbrM2903_10 LN:695527
Program version:
picard-2.6.0
java version "1.8.0_101"