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Old 01-08-2016, 12:00 AM   #1
ea11
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Location: Southampton

Join Date: Jun 2015
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Default Picard tools error

Hi,

I am attempting to use CollectRnaSeqMetrics from picard tools on my BAM files but I am not being successful with it for some reason. Below is the code that I am using:

PHP Code:
java -jar /local/software/picard-tools/1.97/jarlib/CollectRnaSeqMetrics.jar REF_FLAT=refFlat.txt STRAND=SECOND_READ_TRANSCRIPTION_STRAND I=Pre_1.bam O=RnaSeqMetrics_1.txt 
The problem is the next line:
PHP Code:
INFO    2016-01-08 08:54:50     CollectRnaSeqMetrics    Loaded 0 genes
So it says that 0 genes are being loaded, but then it goes on to process the lines in the BAM file and the resulting output says all the reads are intergenic as it is not finding any genes. I have tried it with several other refFlat files, but cant seem to get the program to load any genes with any of the files.

I downloaded the refFlat file from UCSC and changed the chromosome names so they match the BAM file

refFlat file:

PHP Code:
HSD3B2  NM_000198       1       +       119415119       119423039       119415419       119422620       4       "119415119,119415330,119419417,119421808,"      "119415202,119415561,119419582,119423039,"
ANKRD65 NM_001243536    1       -       1418419 1421444 1419541 1421005 3       "1418419,1420815,1421132,"      "1419549,1421005,1421444,"
ANKRD65 NM_001243535    1       -       1418419 1421444 1419233 1421005 3       "1418419,1420796,1421132,"      "1419549,1421005,1421444,"
PDIK1L  NM_001243533    1       +       26111776        26125548        26114308        26122577        4       "26111776,26112459,26114291,26121836,"  "26111915,26112650,26114593,26125548,"
PDIK1L  NM_001243532    1       +       26111164        26125548        26114308        26122577        3       "26111164,26114291,26121836,"   "26111420,26114593,26125548,"
SLC25A24        NM_013386       1       -       108134721       108200358       108136652       108200138       10      "108134721,108139057,108143542,108148278,108154982,108157461,108161181,108181940,108185827,108199955,"    "108136837,108139208,108143710,108148386,108155135,108157620,108161293,108182028,108185954,108200358,"
SLC27A3 NM_001317929    1       +       153775291       153780157       153775356       153780002       10      "153775291,153776517,153777061,153777760,153778160,153778462,153778686,153779113,153779342,153779930,"    "153776164,153776727,153777220,153777885,153778355,153778553,153778885,153779211,153779473,153780157,"
ALG6    NM_013339       1       +       63367589        63438562        63370977        63437020        15      "63367589,63370770,63396512,63402253,63404452,63406316,63407061,63411145,63411925,63414060,63415872,63419369,63428732,63428927,63436822," "63367687,63371059,63396597,63402343,63404541,63406399,63407126,63411331,63412061,63414146,63415957,63419440,63428801,63429126,63438562,"
TPM3    NR_103461       1       -       154155303       154183249       154183249       154183249       8       "154155303,154170399,154170648,154172028,154172907,154173083,154176114,154182987,"        "154157721,154170465,154170711,154172104,154172978,154173201,154176248,154183249,"
NR1I3   NM_001077481    1       -       161229665       161238210       161229796       161236565       8       "161229665,161230812,161231328,161232806,161233168,161235846,161236458,161238040,"        "161229926,161230933,161231474,161232946,161233338,161235977,161236598,161238210," 
BAM file:

PHP Code:
HWI-ST1041:151:C7BJEACXX:1:1101:1127:17612      83      8       125231917       50      35M     =       125231901       -51     TCAGTATATGCTGGCATACTGAATAAATAAATTAA     4D@:4>>:9?@@BBA>>@>>AAB;>5>5;3>1;5,       AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:35 YT:Z:UU NH:i:1  XS:A:+
HWI-ST1041:151:C7BJEACXX:1:1101:1127:17612      163     8       125231901       50      51M     =       125231917       51      TGGTACATGGTAGTAGTCAGTATATGCTGGCATACTGAATAAATAAATTAA       0-3:-;)=>@:99=>)9<4;@8@4@????<?<?48;>>>??8=?<.===<>     AS:i:-2 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:15T35      YT:Z:UU NH:i:1  XS:A:+
HWI-ST1041:151:C7BJEACXX:1:1101:1128:82805      83      5       81259354        50      43M     =       81259277        -120    CTTTCTCTATCTACAATATAAAAATAAGAAAAAAATTAAGTTT     AB=A?0???14=ABBAA@:BBBBBABB?ACB=CCA<+A<2++1       AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:43 YT:Z:UU NH:i:1  XS:A:+
HWI-ST1041:151:C7BJEACXX:1:1101:1128:82805      163     5       81259277        50      100M    =       81259354        120     CCTGCTGGGCCAGGATCAGCTCAGATGGGACTGCTTTGTGGAGGAAGAAAACATCACTGCCCATTCCTTAACCCTTACTTTCTCTATCTACAATATAAAA      0;9?(==???>9>?8??>?<?@?<>?@):.=?????=>??9)9=-39.7<>5>=>??>;8>;>>?@@@>).8=?;>==>????=>>>??==5;:3==:;;    AS:i:-4 XN:i:0  XM:i:2    XO:i:0  XG:i:0  NM:i:2  MD:Z:4A22C72    YT:Z:UU NH:i:1  XS:A:+
Just for reference, the fastq files were paired end and aligned using tophat2 using GRCh38 index.
If anyone can help me with this, it would be much appreciated,
Thanks

Last edited by ea11; 01-08-2016 at 12:02 AM.
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