Hi All,
I just got a MiSeq run back from our core and I am trying to demultiplex everything. However, I have received no instructions as to how I do this, so I am a bit in the dark. The run was 151bp paired-end and the first eight bases make up the barcode.
I have been doing the following:
1. Extract Illumina barcodes using the following command:
The 'extract.txt' file containing the barcodes look like this:
The output from this creates 12 s_1_00xx_barcode.txt files as expected - e.g.:
However, this doesn't look right as all the 12 files look very similar to this with only the first barcode (aggtaagg) displayed in the third column? I'm wondering whether my READ_STRUCTURE=8B143T isn't correct or whether the barcode file isn't read correctly?
2. Alternatively I have given all the barcodes in the command:
But now the output looks different:
This also doesn't look correct.
When I run the IlluminaBasecallsToSam command:
I get a bunch of errors. For number 1) above I get the following:
For number 2) above I get this:
My barcode.txt input for the IlluminaBasecallsToSam.jar command looks like this:
Any idea what is going on here? Any help would be MUCH appreciated - I'm lost!
I just got a MiSeq run back from our core and I am trying to demultiplex everything. However, I have received no instructions as to how I do this, so I am a bit in the dark. The run was 151bp paired-end and the first eight bases make up the barcode.
I have been doing the following:
1. Extract Illumina barcodes using the following command:
Code:
java -Xmx2g -jar /usr/local/bin/picard/ExtractIlluminaBarcodes.jar BASECALLS_DIR=/run/Data/Intensities/BaseCalls LANE=1 BARCODE_FILE=~/Desktop/extract.txt READ_STRUCTURE=8B143T METRICS_FILE=metrics NUM_PROCESSORS=6
Code:
barcode_sequence_1 barcode_sequence_2 barcode_sequence_3 barcode_sequence_4 barcode_sequence_5 barcode_sequence_6 barcode_sequence_7 barcode_sequence_8 barcode_sequence_9 barcode_sequence_10 AGGTAAGG TGCTCGAC GCCTAGCC TTGAGCCT TATCCAGG TGCTGCTG GACCTAAC CTACCAGG CTGCGGAT GTAACATC
Code:
.CTGATTT N aggtaagg 6 9 .TCCTCCT N 8 0 .CCTGCTT N aggtaagg 6 9 CCCACACC N aggtaagg 7 9 CTCGCTCC N 8 0 CCTCAGCC N aggtaagg 7 9 TGCCAGTA N aggtaagg 6 9 CTTTTGTT N aggtaagg 7 9 CGACTCCT N aggtaagg 7 9 GTTACGCG N aggtaagg 7 9
2. Alternatively I have given all the barcodes in the command:
Code:
java -Xmx2g -jar /usr/local/bin/picard/ExtractIlluminaBarcodes.jar BASECALLS_DIR=/run/Data/Intensities/BaseCalls LANE=1 BARCODE=AGGTAAGG BARCODE=TGCTCGAC BARCODE=GCCTAGCC BARCODE=TTGAGCCT BARCODE=TATCCAGG BARCODE=TGCTGCTG BARCODE=GACCTAAC BARCODE=CTACCAGG BARCODE=CTGCGGAT BARCODE=GTAACATC READ_STRUCTURE=8B143T METRICS_FILE=metrics NUM_PROCESSORS=6
Code:
.TCCTCCT N ttgagcct 3 4 .CCTGCTT N tgctgctg 2 4 CCCACACC N gcctagcc 4 4 CTCGCTCC N tgctcgac 5 5 CCTCAGCC N gcctagcc 3 5 TGCCAGTA N tgctcgac 4 4 CTTTTGTT N ctgcggat 4 6 CGACTCCT N ttgagcct 5 5 GTTACGCG N gtaacatc 4 5 CCCCCATT N ctaccagg 4 5 CTAACACG N ctaccagg 2 3
When I run the IlluminaBasecallsToSam command:
Code:
java -Xmx2g -jar /usr/local/bin/picard/IlluminaBasecallsToSam.jar BASECALLS_DIR=/run/Data/Intensities/BaseCalls LANE=1 RUN_BARCODE=120423_MiSeq SEQUENCING_CENTER=Broad BARCODE_PARAMS=~/Desktop/barcodes.txt READ_STRUCTURE=8B143T NUM_PROCESSORS=6 ADAPTERS_TO_CHECK=PAIRED_END
Code:
Picard version: 1.67(1190) INFO 2012-04-25 21:09:47 IlluminaBasecallsToSam READ STRUCTURE IS 8B143T INFO 2012-04-25 21:09:47 IlluminaBasecallsToSam Creating 6 TileProcessors. Before explicit GC, Runtime.totalMemory()=85000192 After explicit GC, Runtime.totalMemory()=85000192 ERROR 2012-04-25 21:09:47 IlluminaBasecallsToSam Exception in TileProcessor net.sf.picard.PicardException: Barcode encountered in that was not specified in BARCODE_PARAMS: null at net.sf.picard.illumina.IlluminaBasecallsToSam$TileProcessor.processTile(IlluminaBasecallsToSam.java:639) at net.sf.picard.illumina.IlluminaBasecallsToSam$TileProcessor.run(IlluminaBasecallsToSam.java:592) at java.lang.Thread.run(Thread.java:680)
Code:
Exception in thread "main" net.sf.picard.PicardException: Could not find a format with available files for the following data types: Position, Barcodes, BaseCalls, QualityScores, PF at net.sf.picard.illumina.parser.IlluminaDataProviderFactory.<init>(IlluminaDataProviderFactory.java:134) at net.sf.picard.illumina.IlluminaBasecallsToSam.doWork(IlluminaBasecallsToSam.java:152) at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:177) at net.sf.picard.illumina.IlluminaBasecallsToSam.main(IlluminaBasecallsToSam.java:377)
Code:
BARCODE OUTPUT SAMPLE_ALIAS LIBRARY_NAME AGGTAAGG Control.bam Control MiSeq_Test TGCTCGAC LASV_90.bam LASV_90 MiSeq_Test GCCTAGCC LASV_241.bam LASV_241 MiSeq_Test TTGAGCCT LASV_245.bam LASV_245 MiSeq_Test TATCCAGG LASV_254.bam LASV_254 MiSeq_Test TGCTGCTG LASV_263.bam LASV_263 MiSeq_Test GACCTAAC LASV_289.bam LASV_289 MiSeq_Test CTACCAGG LASV_291.bam LASV_291 MiSeq_Test CTGCGGAT LASV_295.bam LASV_295 MiSeq_Test GTAACATC LASV_309.bam LASV_309 MiSeq_Test
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