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Demultiplexing problem
Hi All,
I have been trying to demultiplex some Illumina (101bp PE, dual barcoded) libraries using Picard - e.g.:
I have done this a million times before, but with these new libraries I keep getting the following error messages:
I have never seen this happen before and I have tried everything (I can think of...) to fix the problem. Basically it appears as if Picard isn’t accepting the dual barcodes and just treats them individually - which is strange, since I have demultiplexed many runs that were dual barcoded. If I just to try to demultiplex samples where both barcodes are different, then everything goes through.
My barcode data files look like this:
Any idea how to fix this? It's driving me potty.
I have been trying to demultiplex some Illumina (101bp PE, dual barcoded) libraries using Picard - e.g.:
Code:
java -Xmx2g -jar /seq/software/picard/1.626/bin/ExtractIlluminaBarcodes.jar BASECALLS_DIR=/analysis/140615/_temp/140615.H9FT9ADXX/Data/Intensities/BaseCalls/ LANE=2 READ_STRUCTURE=101T8B101T BARCODE_FILE=/analysis/140615/barcodeData.2 METRICS_FILE=/analysis/140615/_logs/barcode.metrics.H9FT9ADXX.2.txt MAX_MISMATCHES=0 MINIMUM_BASE_QUALITY=25 NUM_PROCESSORS=1
Code:
Barcode AGGCAGAA specified more than once in /analysis/140615/barcodeData.2 Barcode CAGAGAGG specified more than once in /analysis/140615/barcodeData.2 Barcode CAGAGAGG specified more than once in /analysis/140615/barcodeData.2 . . .
My barcode data files look like this:
Code:
barcode_name library_name barcode_sequence_1 barcode_sequence_2 H9FT9ADXX-EM-096_NuGEN EM-096_NuGEN AGGCAGAA TATCCTCT H9FT9ADXX-EM-098_NuGEN EM-098_NuGEN AGGCAGAA AGAGTAGA H9FT9ADXX-EM-095B_NuGEN EM-095B_NuGEN CAGAGAGG TAGATCGC H9FT9ADXX-G3691-1_NuGEN G3691-1_NuGEN CAGAGAGG CTCTCTAT H9FT9ADXX-K562B_NuGEN K562B_NuGEN CAGAGAGG CTAAGCCT H9FT9ADXX-EM-095_NuGEN EM-095_NuGEN CTCTCTAC CTAAGCCT H9FT9ADXX-G3670-1_NuGEN G3670-1_NuGEN CTCTCTAC TAGATCGC H9FT9ADXX-G3679-1_NuGEN G3679-1_NuGEN CTCTCTAC TATCCTCT H9FT9ADXX-G3680-1_NuGEN G3680-1_NuGEN CTCTCTAC AGAGTAGA H9FT9ADXX-G3681-1_NuGEN G3681-1_NuGEN CTCTCTAC GTAAGGAG H9FT9ADXX-G3682-1_NuGEN G3682-1_NuGEN CTCTCTAC ACTGCATA H9FT9ADXX-G3683-1_NuGEN G3683-1_NuGEN CTCTCTAC AAGGAGTA H9FT9ADXX-G3686-1_NuGEN G3686-1_NuGEN GGACTCCT ACTGCATA H9FT9ADXX-G3676-1_NuGEN G3676-1_NuGEN TAGGCATG AAGGAGTA H9FT9ADXX-G3676-2_NuGEN G3676-2_NuGEN TAGGCATG CTAAGCCT H9FT9ADXX-G3677-1_NuGEN G3677-1_NuGEN TAGGCATG GTAAGGAG H9FT9ADXX-G3677-2_NuGEN G3677-2_NuGEN TAGGCATG ACTGCATA H9FT9ADXX-G3687-1_NuGEN G3687-1_NuGEN TAGGCATG TAGATCGC
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